Ex vivo expansion of hematopoietic stem cells (HSC) requires the maintenance of a stemness state while cells are proliferating. This can be achieved via exposure to UM171 which leads to the degradation of chromatin modifiers and prevents the loss of key epigenetic marks. However, the chromatin landscape varies across populations within the hematopoietic system and the effect of UM171 on self-renewal and differentiation potential of different hematopoietic progenitor cells is less characterized. To address this, we use the CellTag barcoding approach to track the fate of individual stem and progenitor cells during in vitro expansion. We show that, in addition to its HSC self-renewing property, UM171 specifically modulates cell fate of a precursor common to erythroid, megakaryocytic, and mast cells in favor of self-renewal and a mast-bias differentiation trajectory. This differentiation bias can be driven by pro-inflammatory signaling pathways that are activated downstream of UM171 and results in an abundant mast cell population that can be transplanted as part of the graft to populate mice tissues in xenotransplantation studies.
© 2024. The Author(s).

Acute myeloid leukemia (AML) is the most common hematological cancer in the adult population worldwide. Approximately 35% of patients with AML present internal tandem duplication (ITD) mutations in the FMS‑like tyrosine kinase 3 (FLT3) receptor associated with poor prognosis, and thus, this receptor is a relevant target for potential therapeutics. Tyrosine kinase inhibitors (TKIs) are used to treat AML; however, their molecular interactions and effects on leukemic cells are poorly understood. The present study aimed to gain insights into the molecular interactions and affinity forces of four TKI drugs (sorafenib, midostaurin, gilteritinib and quizartinib) with the wild‑type (WT)‑FLT3 and ITD‑mutated (ITD‑FLT3) structural models of FLT3, in its inactive aspartic acid‑phenylalanine‑glycine motif (DFG‑out) and active aspartic acid‑phenylalanine‑glycine motif (DFG‑in) conformations. Furthermore, the present study evaluated the effects of the second‑generation TKIs gilteritinib and quizartinib on cancer cell viability, apoptosis and proliferation in the MV4‑11 (ITD‑FLT3) and HL60 (WT‑FLT3) AML cell lines. Peripheral blood mononuclear cells (PBMCs) from a healthy volunteer were included as an FLT3‑negative group. Molecular docking analysis indicated higher affinities of second‑generation TKIs for WT‑FLT3/DFG‑out and WT‑FLT3/DFG‑in compared with those of the first‑generation TKIs. However, the ITD mutation changed the affinity of all TKIs. The in vitro data supported the in silico predictions: MV4‑11 cells presented high selective sensibility to gilteritinib and quizartinib compared with the HL60 cells, whereas the drugs had no effect on PBMCs. Thus, the current study presented novel information about molecular interactions between the FLT3 receptors (WT or ITD‑mutated) and some of their inhibitors. It also paves the way for the search for novel inhibitory molecules with potential use against AML.

Umbilical cord blood (CB) is a donor source for hematopoietic cell therapies. Understanding what drives hematopoietic stem and progenitor cell function is critical to our understanding of the usage of CB in hematopoietic cell therapies. Here, we describe how to isolate and analyze the function of human hematopoietic cells from umbilical CB. This protocol demonstrates assays that measure phenotypic properties and hematopoietic cell potency. For complete details on the use and execution of this protocol, please refer to Broxmeyer et al.1.
Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Background: Patients with acute myeloid leukemia (AML) carrying Fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) mutations often face a poor prognosis, high risk of relapse, and short overall survival. While some FLT3 small molecule inhibitors have been used clinically, challenges such as short efficacy, poor specificity, and resistance persist. Proteolytic targeting chimera (PROTAC), with its lower ligand affinity requirement for target proteins, offers higher targeting capability, and the minimal amount of PROTAC can rapidly degrade proteins. This technology may address issues of poor specificity or resistance seen with small molecule inhibitors. Methods Gilteritinib was used as the ligand for the target protein FLT3-ITD, and different E3 ligase ligands were connected to synthesize several series of PROTAC targeting FLT3-ITD. Results Through in vitro and in vivo screening and structural optimization, PROTAC Z29 linked to VHL E3 ligase ligand, the optimal lead compound, was obtained. Kinase screening showed that the synthesized PROTAC Z29 exhibited better specificity and targeting ability than existing small molecule inhibitors. In vitro , Z29 significantly inhibited the proliferation of FLT3-ITD + MOLM13 and MV-4-11 cells and induced FLT3 protein degradation through the proteasome pathway at the nanomolar level. In vivo , Z29 significantly inhibited tumor growth rate at a dose of 30 mg/kg in subcutaneous xenograft mice while maintaining the stable body weight of mice. Compared to Gilteritinib, Z29 , when used in combination with Venetoclax, demonstrated a higher synergy score in MOLM13 and MV-4-11 cells. We also verified Z29 's minimal impact on platelets in a patient-derived xenografts (PDX) model, which may be due to the minimal expression of VHL ligase in platelets. The combination of Z29 and Venetoclax showed better anti-tumor effects and lower platelet toxicity and hepatic toxicity. Conclusion PROTAC technology can enhance drug targeting specificity. The rapid degradation ability and higher targeting specificity of FLT3-ITD PROTAC can mitigate the platelet toxicity of small molecule inhibitors, ensuring safety and efficacy in monotherapy and combination therapy. These findings establish a solid foundation for FLT3-ITD-PROTAC as an effective strategy for the treatment of patients with FLT3-ITD mutation.

Umbilical cord blood transplantation is a life-saving treatment for malignant and non-malignant hematologic disorders. It remains unclear how long cryopreserved units remain functional, and the length of cryopreservation is often used as a criterion to exclude older units. We demonstrate that long-term cryopreserved cord blood retains similar numbers of hematopoietic stem and progenitor cells compared with fresh and recently cryopreserved cord blood units. Long-term cryopreserved units contain highly functional cells, yielding robust engraftment in mouse transplantation models. We also leverage differences between units to examine gene programs associated with better engraftment. Transcriptomic analyses reveal that gene programs associated with lineage determination and oxidative stress are enriched in high engrafting cord blood, revealing potential molecular markers to be used as potency markers for cord blood unit selection regardless of length of cryopreservation. In summary, cord blood units cryopreserved for extended periods retain engrafting potential and can potentially be used for patient treatment.
Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.