UBTF-TD Expression Is Necessary and Sufficient for Myeloid Cell Expansion

Children with acute myeloid leukemia (AML) have a high relapse rate and dismal overall survival. This can be attributed to a limited number of targeted therapies and a poor understanding of the molecular pathogenesis of AML in childhood. Our group recently identified a subtype of pediatric AML defined by recurrent somatic in-frame tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) (Umeda et al., 2022). UBTF-TDs are primarily found in children (~4% of cases at diagnosis), enriched in relapse (~9% of relapse cases), and associated with minimal residual disease (MRD) positivity. UBTF-TD mutations are mutually exclusive with known subtype-defining oncoproteins yet are associated with normal karyotype or trisomy 8 and tend to co-occur with WT1 mutations and FLT3-ITD. Wild-type (WT) UBTF is involved in nucleosome maintenance, chromatin remodeling, and rRNA biogenesis, but the consequences of the tandem duplications on UBTF function, and possibly leukemogenesis, are unknown. We found that expression of UBTF-TD in cord-blood CD34+ (cbCD34+) cells results in increased replating capacity compared to WT-UBTF overexpression as measured by colony forming unit (CFU) assay (UBTF-TD > 4 rounds, WT-UBTF < 2 rounds, q-value < 0.001) as well as increased cellular proliferation. UBTF-TD expression also maintains expression of the stem-cell marker CD117, decreases terminally differentiated myeloid cells (CD11b), and activates the HOXB gene cluster—recapitulating the transcriptional signature observed in patients with UBTF-TD alterations. We, therefore, hypothesized that UBTF-TD expression is a leukemia-initiating event and that leukemogenesis is dependent on UBTF-TD expression.

To assess the leukemic potential of UBTF-TD in vivo, we expressed UBTF-TD in cbCD34+ cells and transplanted them into humanized NSG-SGM3 mice (hSCF hGM-CSF, IL-3) to assess the leukemic potential of UBTF-TD. We observed a rapid in vivo expansion of UBTF-TD expressing cells with an average latency of 11.7 weeks. Peripheral blood immunophenotyping of these mice identified an expansion of myeloid cells (CD33+, CD11b+) and splenomegaly compared to wild-type controls (p-value = 0.0002). These data suggest that UBTF-TD is sufficient to promote myeloid expansion in vivo.

To test whether human cbCD34+ myeloid cell expansion is dependent on UBTF-TD expression, we developed an in vitro inducible degradation model by integrating a targetable protein (FKBP12^F36V) into our standard lentiviral overexpression vector. This system (dTAG) allows for rapid and direct control of UBTF-TD protein expression. Degradation is achieved by treating these cells with small molecules (i.e., dTAG-13) that recruit the FKBP12^F36V-HA-UBTF-TD fusion protein to E3-ubiquitin ligase complex for ubiquitination and subsequent proteasome degradation. Our data shows that the FKBP12^F36V tag has no effect on proliferation and replating capacity of UBTF-TD cbCD34+ cells. Treatment with 1 uM dTAG-13 quickly depleted FKBP12^F36V-HA-UBTF-TD protein within 1 hour of treatment (fold change when compared to DMSO > 75%); this protein was completely undetectable by western blot after 4hrs. These cells displayed a reduction of CD117 surface marker expression (p-value < 0.0001) as measured by flow cytometry—highlighting a loss in stemness. Furthermore, long-term depletion of UBTF-TD resulted in decreased proliferation (p-value < 0.0001) and cell viability (p-value < 0.0001). This coincided with an increase in apoptosis as measured by Annexin V staining (p-value < 0.0001).

Collectively, our data suggest that UBTF-TD expression is sufficient to promote myeloid cell expansion and a leukemic expression profile. This study will help guide therapeutic development in AML as our data shows that directly targeting UBTF-TD for degradation is sufficient to reverse UBTF-TD myeloid cell expansion.

Umeda, M., Ma, J., Huang, B. J., Hagiwara, K., Westover, T., Abdelhamed, S., Klco, J. M. (2022). Integrated genomic analysis identifies UBTF tandem duplications as a recurrent lesion in pediatric acute myeloid leukemia. Blood Cancer Discov. doi:10.1158/2643-3230.BCD-21-0160