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Therapy-related myeloid neoplasms (tMNs) are severe adverse events that can occur after treatment with autologous hematopoietic stem cell transplantation (ASCT). This study aimed to investigate the development of tMNs following ASCT at the molecular level by whole-exome sequencing (WES) and targeted deep sequencing (TDS) in sequential (pre-) tMN samples. WES identified a significantly higher number of mutations in tMNs as compared with de novo myelodysplastic syndrome (MDS) (median 27 vs 12 mutations; = .001). The mutations found in tMNs did not carry a clear aging-signature, unlike the mutations found in de novo MDS, indicating a different mutational mechanism. In some patients, tMN mutations were identified in both myeloid and T cells, suggesting that tMNs may originate from early hematopoietic stem cells (HSCs). However, the mutational spectra of tMNs and the preceding malignancies did not overlap, excluding common ancestry for these malignancies. By use of TDS, tMN mutations were identified at low variant allele frequencies (VAFs) in transplant material in 70% of the patients with tMNs. Reconstruction of clonal patterns based on VAFs revealed that premalignant clones can be present more than 7 years preceding a tMN diagnosis, a finding that was confirmed by immunohistochemistry on bone marrow biopsies. Our results indicate that tMN development after ASCT originates in HSCs bearing (pre-)tMN mutations that are present years before disease onset and that post-ASCT treatment can influence the selection of these clones. Early detection of premalignant clones and monitoring of their evolutionary trajectory may help to predict the development of tMNs and guide early intervention in the future.

As the transcriptional coactivator CITED2 (CBP/p300-interacting-transactivator-with-an ED-rich-tail 2) can be overexpressed in acute myeloid leukemia (AML) cells, we analyzed the consequences of high CITED2 expression in normal and AML cells. CITED2 overexpression in normal CD34(+) cells resulted in enhanced hematopoietic stem and progenitor cell (HSPC) output in vitro, as well as in better hematopoietic stem cell (HSC) engraftability in NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice. This was because of an enhanced quiescence and maintenance of CD34(+)CD38(-) HSCs, due in part to an increased expression of the cyclin-dependent kinase inhibitor CDKN1A. We demonstrated that PU.1 is a critical regulator of CITED2, as PU.1 repressed CITED2 expression in a DNA methyltransferase 3A/B (DNMT3A/B)-dependent manner in normal CD34(+) cells. CD34(+) cells from a subset of AML patients displayed higher expression levels of CITED2 as compared with normal CD34(+) HSPCs, and knockdown of CITED2 in AML CD34(+) cells led to a loss of long-term expansion, both in vitro and in vivo. The higher CITED2 expression resulted from reduced PU.1 activity and/or dysfunction of mutated DNMT3A/B. Collectively, our data demonstrate that increased CITED2 expression results in better HSC maintenance. In concert with low PU.1 levels, this could result in a perturbed myeloid differentiation program that contributes to leukemia maintenance.