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We examined the role of SLFN12, a member of the Schlafen (SLFN) family of interferon-regulated genes and proteins in leukemogenesis, and its potential as a therapeutic target in acute myeloid leukemia (AML). We explored the effects of velcrins, a class of small molecules able to modulate SLFN12 biological activity, on AML cells. Velcrin treatment of AML cells stabilized SLFN12 and promoted SLFN12 complex formation with phosphodiesterase 3A or phosphodiesterase 3B. Such effects were associated with growth-inhibitory and proapoptotic responses, as well as potent suppressive effects on leukemic cell growth. In addition, velcrin treatment suppressed clonogenic capacity of primitive leukemic progenitors and significantly extended survival in a mouse AML xenograft model. Taken together, these findings establish an important role of SLFN12 in leukemogenesis and raise the potential for the use of velcrins as a therapeutic strategy for AML. Significance: Our studies identify SLFN12 as a potential target in AML with important clinical–translational implications.

As a critical mitotic regulator, Aurora kinase A (AURKA) is aberrantly activated in a wide range of cancers. Therapeutic targeting of AUKRA is a promising strategy for the treatment of solid tumors. In this study, we evaluated the preclinical characteristics of JAB-2485, a small-molecule inhibitor of AURKA currently in Phase I/IIa clinical trial in the US ( NCT05490472 ). Biochemical studies demonstrated that JAB-2485 is potent and highly selective on AURKA, with subnanomolar IC 50 and around 1500-fold selectivity over AURKB or AURKC. In addition, JAB-2485 exhibited favorable pharmacokinetic properties featured by low clearance and good bioavailability, strong dose–response relationship, as well as low risk for hematotoxicity and off-target liability. As a single agent, JAB-2485 effectively induced G2/M cell cycle arrest and apoptosis and inhibited the proliferation of small cell lung cancer, triple-negative breast cancer, and neuroblastoma cells. Furthermore, JAB-2485 exhibited robust in vivo antitumor activity both as monotherapy and in combination with chemotherapies or the bromodomain inhibitor JAB-8263 in xenograft models of various cancer types. Together, these encouraging preclinical data provide a strong basis for safety and efficacy evaluations of JAB-2485 in the clinical setting.

PURPOSE Targeting nonspecific, tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However, decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here, we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (Kd {\textless} 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity, preservation of a central memory phenotype, and significantly improved in vivo antitumor function, while preserving their ability to discriminate target antigen density. CONCLUSIONS A combinatorial costimulatory design allows the use of very low-affinity binding domains (Kd {\textless} 1 mumol/L) for the construction of safe but also optimally effective CAR-T cells. Thus, very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs.