Mike Y. Zhong

Bruton tyrosine kinase inhibition is a novel therapeutic strategy targeting tumor in the bone marrow microenvironment in multiple myeloma

Bruton tyrosine kinase (Btk) has a well-defined role in B-cell development, whereas its expression in osteoclasts (OCs) further suggests a role in osteoclastogenesis. Here we investigated effects of PCI-32765, an oral and selective Btk inhibitor, on osteoclastogenesis as well as on multiple myeloma (MM) growth within the BM microenvironment. PCI-32765 blocked RANKL/M-CSF-induced phosphorylation of Btk and downstream PLC-γ2 in OCs, resulting in diminished TRAP5b (ED50 = 17 nM) and bone resorption activity. PCI-32765 also inhibited secretion of multiple cytokines and chemokines from OC and BM stromal cell cultures from both normal donors (ED50 = 0.5 nM) and MM patients. It decreased SDF-1-induced migration of MM cells, and down-regulated MIP1-α/CCL3 in MM cells. It also blocked MM cell growth and survival triggered by IL-6 or coculture with BM stromal cells or OCs in vitro. Importantly, PCI-32765 treatment significantly inhibits in vivo MM cell growth (P < .03) and MM cell-induced osteolysis of implanted human bone chips in SCID mice. Moreover, PCI-32765 prevents in vitro colony formation by stem-like cells from MM patients. Together, these results delineate functional sequelae of Btk activation mediating osteolysis and growth of MM cells, supporting evaluation of PCI-32765 as a novel therapeutic in MM.

Novel anti-B-cell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multiple myeloma

B-cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies. We here show that BCMA is universally expressed on the MM cell surface and determine specific anti-MM activity of J6M0-mcMMAF (GSK2857916), a novel humanized and afucosylated antagonistic anti-BCMA antibody-drug conjugate via a noncleavable linker. J6M0-mcMMAF specifically blocks cell growth via G2/M arrest and induces caspase 3-dependent apoptosis in MM cells, alone and in coculture with bone marrow stromal cells or various effector cells. It strongly inhibits colony formation by MM cells while sparing surrounding BCMA-negative normal cells. J6M0-mcMMAF significantly induces effector cell-mediated lysis against allogeneic or autologous patient MM cells, with increased potency and efficacy compared with the wild-type J6M0 without Fc enhancement. The antibody-dependent cell-mediated cytotoxicity and apoptotic activity of J6M0-mcMMAF is further enhanced by lenalidomide. Importantly, J6M0-mcMMAF rapidly eliminates myeloma cells in subcutaneous and disseminated mouse models, and mice remain tumor-free up to 3.5 months. Furthermore, J6M0-mcMMAF recruits macrophages and mediates antibody-dependent cellular phagocytosis of MM cells. Together, these results demonstrate that GSK2857916 has potent and selective anti-MM activities via multiple cytotoxic mechanisms, providing a promising next-generation immunotherapeutic in this cancer.

Intracellular NAD+ depletion enhances bortezomib-induced anti-myeloma activity

We recently demonstrated that Nicotinamide phosphoribosyltransferase (Nampt) inhibition depletes intracellular NAD⁺ content leading, to autophagic multiple myeloma (MM) cell death. Bortezomib has remarkably improved MM patient outcome, but dose-limiting toxicities and development of resistance limit its long-term utility. Here we observed higher Nampt messenger RNA levels in bortezomib-resistant patient MM cells, which correlated with decreased overall survival. We demonstrated that combining the NAD⁺ depleting agent FK866 with bortezomib induces synergistic anti-MM cell death and overcomes bortezomib resistance. This effect is associated with (1) activation of caspase-8, caspase-9, caspase-3, poly (ADP-ribose) polymerase, and downregulation of Mcl-1; (2) enhanced intracellular NAD⁺ depletion; (3) inhibition of chymotrypsin-like, caspase-like, and trypsin-like proteasome activities; (4) inhibition of nuclear factor κB signaling; and (5) inhibition of angiogenesis. Furthermore, Nampt knockdown significantly enhances the anti-MM effect of bortezomib, which can be rescued by ectopically overexpressing Nampt. In a murine xenograft MM model, low-dose combination FK866 and Bortezomib is well tolerated, significantly inhibits tumor growth, and prolongs host survival. Taken together, these findings indicate that intracellular NAD⁺ level represents a major determinant in the ability of bortezomib to induce apoptosis in MM cells and provide proof of concept for the combination with FK866 as a new strategy to enhance sensitivity or overcome resistance to bortezomib.

APRIL and BCMA promote human multiple myeloma growth and immunosuppression in the bone marrow microenvironment.

Here we show that overexpression or activation of B-cell maturation antigen (BCMA) by its ligand, a proliferation-inducing ligand (APRIL), promotes human multiple myeloma (MM) progression in vivo. BCMA downregulation strongly decreases viability and MM colony formation; conversely, BCMA overexpression augments MM cell growth and survival via induction of protein kinase B (AKT), MAPK, and nuclear factor (NF)-κB signaling cascades. Importantly, BCMA promotes in vivo growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly increased CD31/microvessel density and vascular endothelial growth factor compared with paired control tumors. These tumors also express increased transcripts crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as genes mediating immune inhibition including programmed death ligand 1, transforming growth factor β, and interleukin 10. These target genes are consistently induced by paracrine APRIL binding to BCMA on MM cells, which is blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells even in the presence of protective bone marrow (BM) myeloid cells including osteoclasts, macrophages, and plasmacytoid dendritic cells. 01A further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and noncanonical NF-κB pathways. Moreover, 01A prevents in vivo MM cell growth within implanted human bone chips in SCID mice. Finally, the effect of 01A on MM cell viability is enhanced by lenalidomide and bortezomib. Taken together, these data delineate new molecular mechanisms of in vivo MM growth and immunosuppression critically dependent on BCMA and APRIL in the BM microenvironment, further supporting targeting this prominent pathway in MM.

Abstract 3283: APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment

Elevated B cell maturation antigen (BCMA) and its ligand a proliferation-inducing ligand (APRIL) may directly advance human multiple myeloma (MM) malignancy in vivo. Here, we first show that BCMA downregulation potently diminishes viability and colony formation of MM cells while BCMA overexpression augments MM cell growth and survival via induction of AKT, MAPK, and NFκB signaling cascades and molecules essential for proliferation and anti-apoptosis. Importantly, BCMA itself forces accelerated tumor growth of xenografted MM cells harboring p53 mutation in mice. BCMA-overexpressing tumors exhibit significantly enhanced CD31/microvessel density and VEGF than paired control tumors. Concurrently, these tumors express increased factors crucial for osteoclast activation, adhesion, and angiogenesis/metastasis, as well as immune inhibition including PD-L1, TGFβ and IL-10 which further triggers 38 IL-10 signaling molecules. In parallel, these identified target genes are induced by paracrine APRIL binding to BCMA in MM cells and they are potently blocked by an antagonistic anti-APRIL monoclonal antibody hAPRIL01A (01A). 01A is cytotoxic against MM cells protected by abnormal bone marrow (BM) myeloid cells, i.e., osteoclasts, macrophages, and plasmacytoid dendritic cells. It further decreases APRIL-induced adhesion and migration of MM cells via blockade of canonical and non-canonical NFκB pathways. It prevents in vivo MM cell growth within implanted human bone chips in SCID mice. 01A-inhibited MM cell viability induced by osteoclasts is further enhanced by lenalidomide and bortezomib. Taken together, these results further characterize new molecular mechanisms of APRIL/BCMA-induced in vivo MM progression and immunosuppressive BM microenvironment, strongly supporting targeting this prominent pathway in MM. Citation Format: Yu-Tzu Tai, Chirag Acharya, Gang An, Michele Moschetta, Mike Y. Zhong, Xiaoyan Feng, Michele Cea, Antonia Cagnetta, Kenneth Wen, Hans van Eenennaam, Andrea van Elsas, Nikhil Munshi, Kenneth Anderson. APRIL/BCMA activation promotes human multiple myeloma progression and further induces immunosuppressive bone marrow microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3283.

Abstract 394: A novel anti-a proliferation-inducing ligand hAPRIL.01A monoclonal antibody targets multiple myeloma cells in the bone marrow microenvironment

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro culture showed that osteoclasts (OC) and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p < 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation activated NF-κB, PI3K/AKT, and ERK1/2 signaling in MM cells. NF-κB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with decreased NF-κB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted proliferation and survival of MM cells, in the presence of OCs or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. This Ab also blocks OC-induced MM cell growth and survival in ongoing SCID-hu model of MM. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment. Note: This abstract was not presented at the meeting. Citation Format: Yu-Tzu Tai, Chirag Acharya, Gang An, Mike Y. Zhong, Xiaoyan Feng, Hua Jiang, Hans van Eenennaam, Andrea van Elsas, Nikhil C. Munshi, Kenneth C. Anderson. A novel anti-a proliferation-inducing ligand hAPRIL.01A monoclonal antibody targets multiple myeloma cells in the bone marrow microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 394. doi:10.1158/1538-7445.AM2015-394

Abstract 972: B-cell maturation antigen (BCMA) activation in human multiple myeloma cells promotes myeloma cell growth and survival in the bone marrow microenvironment via upregulated MCL-1 and NFκB signaling

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA B cell maturation antigen (BCMA), a member of the tumor necrosis factor receptor superfamily (TNFRSF17), is selectively induced during plasma cell (PC) differentiation and is commonly expressed at high levels in malignant PCs. Using real time RT-PCR, we here first showed that BCMA mRNA was upregulated in CD138+ PCs from MM patients compared to normal healthy donors, consistent with high and restrictive BCMA expression in PCs but not normal tissues by gene expression profiling and immunohistochemistry in previously published reports. As a specific MM antigen, BCMA is universally expressed on the MM cell surface, confirmed by CD38+BCMA+ dual immunofluorescence staining. We next found that plasmacytoid dendritic cells (pDC), which support MM cell growth, survival, and drug resistance in the bone marrow (BM) microenvironment, have detectable BCMA mRNA at significantly (9-50-fold) lower levels than CD138+ plasma PCs (p<0.005 for each paired sample) from either MM patients or normal donors. Interestingly, as seen in CD138+ PCs, BCMA transcript is considerably elevated in pDC from MM patients vs. normal donors (p<0.03). In contrast, BCMA is hardly detectable in CD138-negative cells from BM aspirates of MM patients. We further define molecular mechanisms of BCMA activation in MM cells in the BM milieu. BCMA shRNA significantly blocks viability of MM cells (RPMI8226, MM1R, H929) and anti-apoptotic protein MCL-1. Conversely, overexpression of BCMA in multiple MM cell lines (RPMI8226, MM1R, H929) by pCMV6/BCMA vector upregulates MCL-1 expression and MIP-1α, as well as NFκB p65 DNA binding activity. Importantly, stimulation of MM cells by APRIL, a cognate ligand for BCMA and mainly secreted by osteoclasts in the BM milieu, induces NFκB DNA binding activity and activates PI3K/AKT and ERK1/2 signaling. APRIL also induces pro-survival/anti-apoptotic targets (BCL2A1, NFκB1, NFκB2) and chemotactic/osteoclast activating factors (MIP-1α and MIP1β) in a dose-dependent manner. Angiogenesis and adhesion/chemoattractant factors (VEGF, IL-8, CXCL10, RANTES) were also significantly induced upon APRIL stimulation. In contrast, BCMA-Fc protein that blocks APRIL binding to BCMA, inhibits secretion of these cytokines/chemokines, indicating specific response of engagement of BCMA by APRIL in BCMA-expressing MM cells. Finally, APRIL induced adhesion and migration of MM cells whereas BCMA-Fc blocked APRIL-induced responses. Together, our results define active APRIL/BCMA signaling cascade in MM in the BM microenvironment, thus providing a niche for MM disease progression. Moreover, these results strongly support rapid bench to bedside translation of the novel antagonistic anti-BCMA antibody drug conjugate (abstract # 14-A-4420-AACR) to treat MM patients. Citation Format: Yu-Tzu Tai, Chirag Acharya, Mike Y. Zhong, Michele Cea, Antonia Cagnetta, Paul Richardson, Nikhil C. Munshi, Kenneth C. Anderson. B-cell maturation antigen (BCMA) activation in human multiple myeloma cells promotes myeloma cell growth and survival in the bone marrow microenvironment via upregulated MCL-1 and NFκB signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 972. doi:10.1158/1538-7445.AM2014-972

Abstract 644: Novel anti-B cell maturation antigen-monomethyl auristatin F antibody-drug conjugate (GSK2857916) induces potent and selective anti-multiple myeloma activity via enhanced effector function and direct tumor cell killing

B cell maturation antigen (BCMA), highly expressed on malignant plasma cells in human multiple myeloma (MM), has not been effectively targeted with therapeutic monoclonal antibodies (mAbs). We here investigated the anti-MM activity of J6M0-mcMMAF (GSK2857916), a humanized and afucosylated anti-BCMA antibody-drug conjugate (ADC) via uncleavable linker. This novel antagonist anti-BCMA antibody shows binding against all CD138-expressing MM cell lines and patient MM cells, confirming universal BCMA expression on the surface of myeloma cells. Real-time qRT-PCR also showed significantly upregulated BCMA mRNA in CD138+ cells purified from MM patients vs. normal donors (p Note: This abstract was not presented at the meeting. Citation Format: Yu-Tzu Tai, Chirag Acharya, Mike Y. Zhong, Michele Cea, Antonia Cagnetta, Patrick A. Mayes, Jenny Craigen, Louise Gliddon, James Smothers, Amanda L. Christie, Andrew L. Kung, Paul Richardson, Nikhil C. Munshi, Kenneth C. Anderson. Novel anti-B cell maturation antigen-monomethyl auristatin F antibody-drug conjugate (GSK2857916) induces potent and selective anti-multiple myeloma activity via enhanced effector function and direct tumor cell killing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 644. doi:10.1158/1538-7445.AM2014-644