The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging designed ankyrin repeat protein (DARPin) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells coexpressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mice models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity toward LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with a high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

TNFR2 is required for RIP1-dependent cell death in human leukemia.

Despite major advances in the treatment of patients with acute lymphoblastic leukemia in the last decades, refractory and/or relapsed disease remains a clinical challenge, and relapsed leukemia patients have an exceedingly dismal prognosis. Dysregulation of apoptotic cell death pathways is a leading cause of drug resistance; thus, alternative cell death mechanisms, such as necroptosis, represent an appealing target for the treatment of high-risk malignancies. We and other investigators have shown that activation of receptor interacting protein kinase 1 (RIP1)-dependent apoptosis and necroptosis by second mitochondria derived activator of caspase mimetics (SMs) is an attractive antileukemic strategy not currently exploited by standard chemotherapy. However, the underlying molecular mechanisms that determine sensitivity to SMs have remained elusive. We show that tumor necrosis factor receptor 2 (TNFR2) messenger RNA expression correlates with sensitivity to SMs in primary human leukemia. Functional genetic experiments using clustered regularly interspaced short palindromic repeats/Cas9 demonstrate that TNFR2 and TNFR1, but not the ligand TNF-α, are essential for the response to SMs, revealing a ligand-independent interplay between TNFR1 and TNFR2 in the induction of RIP1-dependent cell death. Further potential TNFR ligands, such as lymphotoxins, were not required for SM sensitivity. Instead, TNFR2 promotes the formation of a RIP1/TNFR1-containing death signaling complex that induces RIP1 phosphorylation and RIP1-dependent apoptosis and necroptosis. Our data reveal an alternative paradigm for TNFR2 function in cell death signaling and provide a rationale to develop strategies for the identification of leukemias with vulnerability to RIP1-dependent cell death for tailored therapeutic interventions.

Repurposing anthelmintic agents to eradicate resistant leukemia.

Despite rapid progress in genomic profiling in acute lymphoblastic leukemia (ALL), identification of actionable targets and prediction of response to drugs remains challenging. To identify specific vulnerabilities in ALL, we performed a drug screen using primary human ALL samples cultured in a model of the bone marrow microenvironment combined with high content image analysis. Among the 2487 FDA-approved compounds tested, anthelmintic agents of the class of macrocyclic lactones exhibited potent anti-leukemia activity, similar to the already known anti-leukemia agents currently used in induction chemotherapy. Ex vivo validation in 55 primary ALL samples of both precursor B cell and T-ALL including refractory relapse cases confirmed strong anti-leukemia activity with IC50 values in the low micromolar range. Anthelmintic agents increased intracellular chloride levels in primary leukemia cells, inducing mitochondrial outer membrane depolarization and cell death. Supporting the notion that simultaneously targeting cell death machineries at different angles may enhance the cell death response, combination of anthelmintic agents with the BCL-2 antagonist navitoclax or with the chemotherapeutic agent dexamethasone showed synergistic activity in primary ALL. These data reveal anti-leukemia activity of anthelmintic agents and support exploiting drug repurposing strategies to identify so far unrecognized anti-cancer agents with potential to eradicate even refractory leukemia.