Targeting CD38 with Daratumumab Suppresses B-Cell Receptor Signaling and Enhances the Activity of Ibrutinib in Waldenstrom Macroglobulinemia Cells

Abstract

Introduction: Waldenstrom macroglobulinemia (WM) is characterized by malignant B-lymphoplasmacytic cells that rely on B-cell receptor (BCR) signaling, which is clinically exploited with ibrutinib (Ibr). Although effective, Ibr tx. mostly results in partial remission of disease. This signifies the need to examine combination tx. strategies that can lead to superior disease eradication and counteract onset of Ibr resistance. WM cells express several surface antigens, including CD38 and Daratumumab (Dara) is a first-in-class monoclonal antibody that binds CD38; inducing an immune effector-mediated cytolytic reaction. CD38 also functions as a molecular hub for integration of signals transmitted through the B-cell receptor complex (BCR) to promote cell proliferation. We hypothesized that cotargeting BTK (with Ibr) and CD38 (with Dara) would induce both an immune-mediated and cell intrinsic mode of death to result in enhanced WM cell kill.Methods: Wildtype WM cell lines (BCWM.1, RPCI.WM1) and ibrutinib-resistant (IR) subclones were used in experiments. CD38 MFI and surface antibody binding (sAbc) was determined by flow cytometry. Ibr (1uM), Dara (0.1ug/mL) or Ibr + Dara-mediated death through antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) was measured using the Calcein AM assay at an effector to target (E:T) ratio of 50:1. Antibody-dependent cellular phagocytosis (ADCP) was assessed using human macrophages at an E:T ratio of 2:1. An IgG1 isotype Ab was used as a control in experiments.Results: CD38 sAbc in BCWM.1 and RPCI-WM1 cells was 4864 and 35892, respectively; consistent with previously noted MFI's (Paulus et al PLOS One, 2016). Specific lysis induced by single agent Dara in wildtype WM cells, showed a mean ADCC of 24.46%, CDC: 20.29% and ADCP: 20.12%. Dara can also induce apoptosis and we noted an average of 33.35% annexin-V/PI+ WM cells after 24h Dara tx. We surmised the anti-WM activity of Dara could be enhanced by Ibr as 1.) the BTK inhibitor has been shown to improve anti-tumor T-cell responses and 2.) the combined effects of each drug could synergistically attenuate BCR-associated signaling. As anticipated, mean ADCC, CDC, ADCP and apoptosis increased to 52.7%, 38.41%, 30.19% and 57.53%, respectively in Ibr + Dara-treated wildtype WM cells. While potential mechanisms of the enhanced immune-mediated specific lysis from the combination tx. are being investigated, we observed cell signaling changes that may account for the increased cell death. As expected, Dara decreased pLyn, pSyk, pBTK, pPLCG2 and pERK as well as pAKT, pmTOR and pS6K levels in WM cells. And these proteins decreased further in Ibr + Dara treated cells. Next, we asked the question whether Dara +/- Ibr would be active in IR WM cells, which had lower CD38 sAbc (mean 8400, range 1024 - 15776). Mean ADCC by Dara in IR WM cells was 19.52%, CDC: 31.85%, ADCP: 27.5% and apoptosis at 31.3%. While ADCC was expectedly lower in IR vs WT cells, CDC and ADCP were significantly higher. We then tested the combination of Ibr + Dara in IR WM cells and noted greater ADCC (63.97%), CDC (60.34%) and apoptosis (60.23%), whereas ADCP did not increase (29.45%). We also analyzed BCR-associated protein expression and noted single agent Dara decreased pBTK, pPLCG2, pERK and pS6K, whereas the effects of Ibr + Dara were more cell line specific. As IR WM subclones are less reliant on BTK/BCR-mediated signaling, cell death from Dara (+/- Ibr) may be due to other mechanisms possibly related to alterations in the mitochondria (intrinsic to IR cells, Paulus et al BCJ 2017).Conclusion: We demonstrate in vitro activity of Dara and Ibr in WM cells, showing that 1.) Dara induces cell death through various mechanisms (ADCC, CDC, ADCP and apoptosis); 2.) while higher CD38 sAbc appears to correlate with greater ADCC, this was not the case for CDC, ADCP or apoptosis; 3.) Dara significantly modulates BCR-associated signaling; an effect preserved in IR WM cells and documented for the first time and 4.) the combination tx. exerts significantly more WM cell death in nearly all the aforementioned assays than either single agent alone and in some cases more so in IR WM cells. With further preclinical results supporting these data, to be shared at the conference, our investigations provide rationale for clinical testing of Ibr + Dara in WM. DisclosuresSher:LAM Therapeutics, Inc: Research Funding. Ansell:Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Celldex: Research Funding; Affimed: Research Funding; Seattle Genetics: Research Funding. Malavasi:Jaansen Pharmaceuticals: Research Funding. Ailawadhi:Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Pharmacyclics: Research Funding; Takeda: Consultancy, Honoraria.