Purpose: Development of B-cell lymphoma 2 (BCL-2)–specific inhibitors poses unique challenges in drug design because of BCL-2 homology domain 3 (BH3) shared homology between BCL-2 family members and the shallow surface of their protein–protein interactions. We report herein discovery and extensive preclinical investigation of lisaftoclax (APG-2575). Experimental Design: Computational modeling was used to design “lead” compounds. Biochemical binding, mitochondrial BH3 profiling, and cell-based viability or apoptosis assays were used to determine the selectivity and potency of BCL-2 inhibitor lisaftoclax. The antitumor effects of lisaftoclax were also evaluated in several xenograft models. Results: Lisaftoclax selectively binds BCL-2 (Ki < 0.1 nmol/L), disrupts BCL-2:BIM complexes, and compromises mitochondrial outer membrane potential, culminating in BAX/BAK-dependent, caspase-mediated apoptosis. Lisaftoclax exerted strong antitumor activity in hematologic cancer cell lines and tumor cells from patients with chronic lymphocytic leukemia, multiple myeloma, or Waldenström macroglobulinemia. After lisaftoclax treatment, prodeath proteins BCL-2‒like protein 11 (BIM) and Noxa increased, and BIM translocated from cytosol to mitochondria. Consistent with these apoptotic activities, lisaftoclax entered malignant cells rapidly, reached plateau in 2 hours, and significantly downregulated mitochondrial respiratory function and ATP production. Furthermore, lisaftoclax inhibited tumor growth in xenograft models, correlating with caspase activation, poly (ADP-ribose) polymerase 1 cleavage, and pharmacokinetics of the compound. Lisaftoclax combined with rituximab or bendamustine/rituximab enhanced antitumor activity in vivo. Conclusions: These findings demonstrate that lisaftoclax is a novel, orally bioavailable BH3 mimetic BCL-2–selective inhibitor with considerable potential for the treatment of certain hematologic malignancies.

Although ibrutinib is highly effective in Waldenstrom macroglobulinemia (WM), no complete remissions in WM patients treated with ibrutinib have been reported to date. Moreover, ibrutinib-resistant disease is being steadily reported and is associated with dismal clinical outcome (overall survival of 2.9–3.1 months). To understand mechanisms of ibrutinib resistance in WM, we established ibrutinib-resistant in vitro models using validated WM cell lines. Characterization of these models revealed the absence of BTK^C481S and CXCR4^WHIM-like mutations. BTK-mediated signaling was found to be highly attenuated accompanied by a shift in PI3K/AKT and apoptosis regulation-associated genes/proteins. Cytotoxicity studies using the AKT inhibitor, MK2206±ibrutinib, and the Bcl-2-specific inhibitor, venetoclax±ibrutinib, demonstrated synergistic loss of cell viability when either MK22016 or venetoclax were used in combination with ibrutinib. Our findings demonstrate that induction of ibrutinib resistance in WM cells can arise independent of BTK^C481S and CXCR4^WHIM-like mutations and sustained pressure from ibrutinib appears to activate compensatory AKT signaling as well as reshuffling of Bcl-2 family proteins for maintenance of cell survival. Combination treatment demonstrated greater (and synergistic) antitumor effect and provides rationale for development of therapeutic strategies encompassing venetoclax+ibrutinib or PI3K/AKT inhibitors+ibrutinib in ibrutinib-resistant WM.

Purpose: This global phase I trial investigated the safety, efficacy, pharmacokinetics, and pharmacodynamics of lisaftoclax (APG-2575), a novel, orally active, potent selective B-cell lymphoma 2 (BCL-2) inhibitor, in patients with relapsed or refractory chronic lymphocytic leukemia or small lymphocytic lymphoma (R/R CLL/SLL) and other hematologic malignancies (HMs). Patients and Methods: Maximum tolerated dose (MTD) and recommended phase II dose were evaluated. Outcome measures were safety and tolerability (primary) and pharmacokinetic variables and antitumor effects (secondary). Pharmacodynamics in patient tumor cells were explored. Results: Among 52 patients receiving lisaftoclax, MTD was not reached. Treatment-emergent adverse events (TEAEs) included diarrhea (48.1%), fatigue (34.6%), nausea (30.8%), anemia and thrombocytopenia (28.8% each), neutropenia (26.9%), constipation (25.0%), vomiting (23.1%), headache (21.2%), peripheral edema and hypokalemia (17.3% each), and arthralgia (15.4%). Grade ≥ 3 hematologic TEAEs included neutropenia (21.2%), thrombocytopenia (13.5%), and anemia (9.6%), none resulting in treatment discontinuation. Clinical pharmacokinetic and pharmacodynamic results demonstrated that lisaftoclax had a limited plasma residence and systemic exposure and elicited rapid clearance of malignant cells. With a median treatment of 15 (range, 6–43) cycles, 14 of 22 efficacy-evaluable patients with R/R CLL/SLL experienced partial responses, for an objective response rate of 63.6% and median time to response of 2 (range, 2–8) cycles. Conclusions: Lisaftoclax was well tolerated, with no evidence of tumor lysis syndrome. Dose-limiting toxicity was not reached at the highest dose level. Lisaftoclax has a unique pharmacokinetic profile compatible with a potentially more convenient daily (vs. weekly) dose ramp-up schedule and induced rapid clinical responses in patients with CLL/SLL, warranting continued clinical investigation.

Patients with chronic lymphocytic leukemia (CLL) are characterized by monoclonal expansion of CD5+CD23+CD27+CD19+κ/λ+ B lymphocytes and are clinically noted to have profound immune suppression. In these patients, it has been recently shown that a subset of B cells possesses regulatory functions and secretes high levels of interleukin 10 (IL-10). Our investigation identified that CLL cells with a CD19+CD24+CD38hi immunophenotype (B regulatory cell [Breg]–like CLL cells) produce high amounts of IL-10 and transforming growth factor β (TGF-β) and are capable of transforming naive T helper cells into CD4+CD25+FoxP3+ T regulatory cells (Tregs) in an IL-10/TGF-β-dependent manner. A strong correlation between the percentage of CD38+ CLL cells and Tregs was observed. CD38hi Tregs comprised more than 50% of Tregs in peripheral blood mononuclear cells (PBMCs) in patients with CLL. Anti-CD38 targeting agents resulted in lethality of both Breg-like CLL and Treg cells via apoptosis. Ex vivo, use of anti-CD38 monoclonal antibody (mAb) therapy was associated with a reduction in IL-10 and CLL patient-derived Tregs, but an increase in interferon-γ and proliferation of cytotoxic CD8+ T cells with an activated phenotype, which showed an improved ability to lyse patient-autologous CLL cells. Finally, effects of anti-CD38 mAb therapy were validated in a CLL–patient-derived xenograft model in vivo, which showed decreased percentage of Bregs, Tregs, and PD1+CD38hiCD8+ T cells, but increased Th17 and CD8+ T cells (vs vehicle). Altogether, our results demonstrate that targeting CD38 in CLL can modulate the tumor microenvironment; skewing T-cell populations from an immunosuppressive to immune-reactive milieu, thus promoting immune reconstitution for enhanced anti-CLL response.

Monoclonal antibodies (mAbs) were initially considered as a possible “magic bullet” for in vivo elimination of tumor cells. mAbs represented the first step: however, as they were murine in nature (the earliest experience on the field), they were considered unfit for human applications. This prompted the development of techniques for cloning the variable regions of conventional murine antibodies, genetically mounted on human IgG. The last step in this years-long process was the design for the preparation of fully human reagents. The choice of the target molecule was also problematic, since cancer-specific targets are quite limited in number. To overcome this obstacle in the planning phases of antibody-mediated therapy, attention was focused on a set of normal molecules, whose quantitative distribution may balance a tissue-dependent generalized expression. The results and clinical success obtained with anti-CD20 mAbs revived interest in this type of strategy. Using multiple myeloma (MM) as a tumor model was challenging first of all because the plasma cells and their neoplastic counterpart eluded the efforts of the Workshop on Differentiation Antigens to find a target molecule exclusively expressed by these cells. For this reason, attention was turned to surface molecules which fulfill the requisites of being reasonably good targets, even if not specifically restricted to tumor cells. In 2009, we proposed CD38 as a MM target in virtue of its expression: it is absent on early hematological progenitors, has variable but generalized limited expression by normal cells, but is extremely high in plasma cells and in myeloma. Further, regulation of its expression appeared to be dependent on a variety of factors, including exposure to all-trans retinoic acid (ATRA), a potent and highly specific inducer of CD38 expression in human promyelocytic leukemia cells that are now approved for in vivo use. This review discusses the history of human CD38, from its initial characterization to its targeting in antibody-mediated therapy of human myeloma.

Purpose: CD38 has emerged as a high-impact therapeutic target in multiple myeloma, with the approval of daratumumab (anti-CD38 mAb). The clinical importance of CD38 in patients with chronic lymphocytic leukemia (CLL) has been known for over 2 decades, although it9s relevance as a therapeutic target in CLL remains understudied. Experimental Design: We investigated the biological effects and antitumor mechanisms engaged by daratumumab in primary CLL cells. Besides its known immune-effector mechanisms (antibody-dependent cell-mediated cytotoxicity, complement-dependent death, and antibody-dependent cellular phagocytosis), we also measured direct apoptotic effects of daratumumab alone or in combination with ibrutinib. In vivo antileukemic activity was assessed in a partially humanized xenograft model. The influence of CD38 on B-cell receptor (BCR) signaling was measured via immunoblotting of Lyn, Syk, BTK, PLCγ2, ERK1/2, and AKT. Results: In addition to immune-effector mechanisms; daratumumab also induced direct apoptosis of primary CLL cells, which was partially dependent on FcγR cross-linking. For the first time, we demonstrated the influence of CD38 on BCR signaling where interference of CD38 downregulated Syk, BTK, PLCγ2, ERK1/2, and AKT; effects that were further enhanced by addition of ibrutinib. In comparison to single-agent treatment, the combination of ibrutinib and daratumumab resulted in significantly enhanced anti-CLL activity in vitro and significantly decreased tumor growth and prolonged survival in the in vivo CLL xenograft model. Conclusions: Overall, our data demonstrate the antitumor mechanisms of daratumumab in CLL; furthermore, we show how cotargeting BTK and CD38 lead to a robust anti-CLL effect, which has clinical implications.