UM171 Expands Immature Bone Marrow CD34+ Cells of Patients with Telomere-Biology Disorder

Introduction: Telomere-biology disorders (TBD) are caused by germline defects in genes involved in telomere maintenance, resulting in excessive telomere shortening and limited cell proliferation. Clinically, TBD corresponds to a spectrum of phenotypes associated with bone marrow failure and lung and liver diseases. It also is associated with a higher predisposition to cancer, including myelodysplastic syndrome and acute myeloid leukemia. Patients with affected hematopoiesis usually present cytopenias and hypocellular marrow with reduced numbers of CD34⁺ cells to maintain adequate hematopoiesis. Treatment options for marrow failure patients are restricted. The most effective approach is allogeneic bone marrow transplantation, but the availability of compatible donors and complications of the conditioning regimen limit it. Alternatively, androgens may alleviate cytopenias, but their long-term effectiveness may be restricted (Townsley et al. 2016; Clé et al. 2023; Pagliuca et al. 2024). There is no evidence that TPO agonists have any effects in these cases. Fares et al. (2014) demonstrated that the small molecule UM171 expands the CD34⁺ cell subpopulations from human umbilical cord blood with superior engraftment potential and better clinical outcomes (Cohen et al. 2022). UM171 is a pyrimido-indole derivative that activates the CRL3^KBTBD4 E3 ubiquitin ligase, targeting the CoREST1 complex and maintaing the epigenetic landscape required for hematopoietic stem cell properties (Chagraoui et al. 2021). Here, we assessed the potential of UM171 to expand the hematopoietic progenitor and stem cell (HPSC) compartment of patients with TBD and marrow failure.

Methods: Bone marrow samples were collected from six patients diagnosed with TBD (four females; median age, 39.5 years; range, 16 to 56 years). All patients had short telomeres and a hypocellular bone marrow. Two patients were diagnosed with aplastic anemia, three with cytopenias, two with pulmonary disease, and one with liver cirrhosis. Heterozygous TERT mutations were found in four patients, TERC mutations in two, RTEL1 in one, and POT1 in one, all in heterozygosis. Bone marrow samples from six healthy controls (all males; median age, 27,5 years; range, 24 to 71 years) were studied as controls. CD34⁺ cells were enriched using immunomagnetic labeling with human CD34 microbeads and a magnetic separator. Cells were cultured for 7 days in ACF medium supplemented with cytokines that support HSC expansion and UM171 or DMSO (control). To assess the capacity of the cultured cells to generate hematopoietic progenitors, 1,000 cells/mL were resuspended in methylcellulose in triplicate. After 14 days, the colonies were counted and classified according to morphology.

Results: Patients had fewer CD34⁺ cells in the bone marrow compared to controls (median, 0.68% vs. 1.69%, respectively; P=0.03). After a 7-day expansion, the percentage of CD34⁺ cells was higher when treated with UM171 (UM171, 56.9 ±5.7% vs. DMSO, 34.9% ±6.5% [median ±standard error] n=6; P=0.003). The cell surface EPCR is a marker for the purification of the HSPC compartment, and the CD34⁺EPCR⁺ subpopulation was increased after UM171 treatment (UM171, 3.35±1.1% vs. DMSO, 0.2% ±0.1%; n=6; P=0.03). UM171-treated cells gave rise to more progenitor cells, as observed by the CFU assay (UM171, 142±18 vs. DMSO, 94%±9; n=4; P=0.01). No significant telomere attrition was observed with the expansion. We also analyzed the appearance of abnormal clones during expansion by NSG for myeloid-malignancy somatic mutations and chromosomal abnormalities by single nucleotide polymorphism array (SNP-array) in CD34⁺ cells from three patients and two healthy controls. No abnormal clones were observed after UM171 expansion.

Conclusions: Our results reveal UM171 as a potent HSPC expander in TBD patients. Our data suggest that UM171 may enhance the ex vivo production of HSPC from TBD patients. These findings contribute the understanding of the effect of UM171 on patient-derived cells.