The MLL-Menin Interaction is a Therapeutic Vulnerability in <em>NUP98</em>-rearranged AML

\ua9 2023 Wolters Kluwer Health. All rights reserved. Chromosomal translocations involving the NUP98 locus are among the most prevalent rearrangements in pediatric acute myeloid leukemia (AML). AML with NUP98 fusions is characterized by high expression of HOXA and MEIS1 genes and is associated with poor clinical outcome. NUP98 fusion proteins are recruited to their target genes by the mixed lineage leukemia (MLL) complex, which involves a direct interaction between MLL and Menin. Here, we show that therapeutic targeting of the Menin-MLL interaction inhibits the propagation of NUP98-rearrranged AML both ex vivo and in vivo. Treatment of primary AML cells with the Menin inhibitor revumenib (SNDX-5613) impairs proliferation and clonogenicity ex vivo in long-term coculture and drives myeloid differentiation. These phenotypic effects are associated with global gene expression changes in primary AML samples that involve the downregulation of many critical NUP98 fusion protein-target genes, such as MEIS1 and CDK6. In addition, Menin inhibition reduces the expression of both wild-type FLT3 and mutated FLT3-ITD, and in combination with FLT3 inhibitor, suppresses patient-derived NUP98-r AML cells in a synergistic manner. Revumenib treatment blocks leukemic engraftment and prevents leukemia-associated death of immunodeficient mice transplanted with NUP98::NSD1 FLT3-ITD-positive patient-derived AML cells. These results demonstrate that NUP98-rearranged AMLs are highly susceptible to inhibition of the MLL-Menin interaction and suggest the inclusion of AML patients harboring NUP98 fusions into the clinical evaluation of Menin inhibitors

Leukemic stem cells activate lineage inappropriate signalling pathways to promote their growth

Acute Myeloid Leukemia (AML) is caused by multiple mutations which dysregulate growth and differentiation of myeloid cells. Cells adopt different gene regulatory networks specific to individual mutations, maintaining a rapidly proliferating blast cell population with fatal consequences for the patient if not treated. The most common treatment option is still chemotherapy which targets such cells. However, patients harbour a population of quiescent leukemic stem cells (LSCs) which can emerge from quiescence to trigger relapse after therapy. The processes that allow such cells to re-grow remain unknown. Here, we examine the well characterised t(8;21) AML sub-type as a model to address this question. Using four primary AML samples and a novel t(8;21) patient-derived xenograft model, we show that t(8;21) LSCs aberrantly activate the VEGF and IL-5 signalling pathways. Both pathways operate within a regulatory circuit consisting of the driver oncoprotein RUNX1::ETO and an AP-1/GATA2 axis allowing LSCs to re-enter the cell cycle while preserving self-renewal capacity

Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion proteins

In an attempt to improve TRAIL's (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor-targeting properties. We studied tumor targeting and apoptosis induction of scFv–scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv–scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL's antitumoral action and to minimize potential unwanted actions on normal tissues

Superior antitumoral activity of dimerized targeted single-chain TRAIL fusion proteins under retention of tumor selectivity

Although targeting of the death receptors (DRs) DR4 and DR5 still appears a suitable antitumoral strategy, the limited clinical responses to recombinant soluble TNF-related apoptosis inducing ligand (TRAIL) necessitate novel reagents with improved apoptotic activity/tumor selectivity. Apoptosis induction by a single-chain TRAIL (scTRAIL) molecule could be enhanced >10-fold by generation of epidermal growth factor receptor (EGFR)-specific scFv-scTRAIL fusion proteins. By forcing dimerization of scFv-scTRAIL based on scFv linker modification, we obtained a targeted scTRAIL composed predominantly of dimers (Db-scTRAIL), exceeding the activity of nontargeted scTRAIL ∼100-fold on Huh-7 hepatocellular and Colo205 colon carcinoma cells. Increased activity of Db-scTRAIL was also demonstrated on target-negative cells, suggesting that, in addition to targeting, oligomerization equivalent to an at least dimeric assembly of standard TRAIL per se enhances apoptosis signaling. In the presence of apoptosis sensitizers, such as the proteasomal inhibitor bortezomib, Db-scTRAIL was effective at picomolar concentrations in vitro (EC50 ∼2 × 10−12 M). Importantly, in vivo, Db-scTRAIL was well tolerated and displayed superior antitumoral activity in mouse xenograft (Colo205) tumor models. Our results show that both targeting and controlled dimerization of scTRAIL fusion proteins provides a strategy to enforce apoptosis induction, together with retained tumor selectivity and good in vivo tolerance