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Hematopoietic stem cell (HSC) quiescence supports lifelong blood regeneration and guards against pre-leukemic clonal expansion. Acute exposure to the pro-inflammatory cytokine interleukin (IL)-1 drives myeloid cell production and HSC cell cycle entry. However, HSC return to a quiescent state suggesting the presence of a ‘braking’ mechanism that limits HSC proliferative capacity during chronic inflammation. To identify mechanism(s) regulating HSC cell cycle activity, we injected mice with IL-1 for 20 days modeling chronic inflammation in vivo. RNA-seq analysis of HSC following IL-1 exposure revealed repression of cell cycle and protein synthesis genes, suggesting the activation of a ‘growth arrest’ gene program. This gene program coincided with increased PU.1 expression, and ChIP-seq analysis identified PU.1 binding on nearly all repressed genes, suggesting PU.1 enforces HSC quiescence during chronic inflammation. Strikingly, HSC from IL-1 treated PU.1-deficient mice exhibited loss of quiescence associated with aberrant myeloid expansion in the bone marrow and spleen. Together, our results suggest PU.1 induction maintains HSC quiescence under chronic inflammatory stress. They also suggest IL-1 may confer a competitive advantage to HSC with impaired PU.1 function, triggering aberrant proliferation and myeloid expansion. Several oncogenic mutations found in acute myelogenous leukemia (AML) impair the expression and/or function of PU.1. As AML pre-dominantly affects elderly individuals and leukemogenesis is often associated with chronic inflammation, our data supports a model where chronic inflammation triggers the selective expansion of HSC harboring oncogenic mutations, leading to a pre-leukemic state. Thus, blockade of inflammation may be a tractable approach to delay and/or prevent leukemia.

Myelodysplastic syndrome (MDS) is a group of heterogeneous disorders caused by ineffective hematopoiesis and characterized by bone marrow dysplasia and cytopenia. Current treatment options for MDS are limited to supportive care, hypomethylating agents, and stem cell transplant. Most patients eventually succumb to the disease or progress to leukemia. Previously, we found that CD123 can be used to delineate MDS stem cells in patients at high risk for MDS and that the CD123-positive population is biologically distinct from normal hematopoietic stem cells. Furthermore, selective targeting of MDS stem cells can dramatically reduce tumor burden in preclinical models. On the basis of these findings, we propose CD123 as a candidate target for chimeric antigen receptor (CAR) T-cell therapy in high-risk MDS patients. To test this concept, we employed a CAR lentiviral vector containing a CD123-specific single-chain variable fragment in combination with the CD28 costimulatory domain, CD3ζ signaling domain, and truncated estimated glomerular filtration rate. Utilizing this system, we illustrate that CD123 CAR can be expressed on both healthy donor and MDS patient-derived T lymphocytes with high efficiency, leading to the successful elimination of MDS stem cells both in vitro and in patient-derived xenografts. These results provide the concept for the use of CD123-targeted CAR T cells as a therapeutic option for patients with MDS.

Ciclopirox, an antifungal agent commonly used for the dermatologic treatment of mycoses, has been shown recently to have antitumor properties. Although the exact mechanism of ciclopirox is unclear, its antitumor activity has been attributed to iron chelation and inhibition of the translation initiation factor eIF5A. In this study, we identify a novel function of ciclopirox in the inhibition of mTOR. As with other mTOR inhibitors, we show that ciclopirox significantly enhances the ability of the established preclinical antileukemia compound, parthenolide, to target acute myeloid leukemia. The combination of parthenolide and ciclopirox demonstrates greater toxicity against acute myeloid leukemia than treatment with either compound alone. We also demonstrate that the ability of ciclopirox to inhibit mTOR is specific to ciclopirox because neither iron chelators nor other eIF5A inhibitors affect mTOR activity, even at high doses. We have thus identified a novel function of ciclopirox that might be important for its antileukemic activity.

Objective Human organic cation transporters (OCTs) play a critical role in the cellular uptake and efflux of endogenous cationic substrates and hydrophilic exogenous xenobiotics. We sought to identify OCT genes preferentially expressed in hematopoietic cells. Materials and Methods We isolated a novel OCT, named OCT6, by data-mining human expressed sequence tag databases for sequences homologous to known OCT genes. We developed a quantitative reverse transcriptase polymerase chain reaction assay to determine the relative expression of this gene in 50 cancer cell lines and in tissues. Results The two highest expressing cell lines were the leukemia cell lines HL-60 and MOLT4. Quantitative reverse transcriptase polymerase chain reaction analysis using a normal tissue cDNA panel demonstrated that this transport gene is highly expressed in testis and fetal liver, with detectable RNA levels in bone marrow and peripheral blood leukocytes. Unlike other OCT genes, RNA levels were not detectable in placenta, liver, or kidney. To further define the expression of OCT6 in hematopoietic tissues, we measured OCT6 RNA levels in sorted peripheral blood cell populations and found a clear enrichment of OCT6-expressing cells in purified CD34 + cells. To determine if OCT6 was highly expressed in leukemias, we examined circulating leukemia cells from 25 patients and found high levels of OCT6 RNA in all specimens in comparison with liver, kidney, and placenta. Conclusion The results demonstrate the existence of a novel OCT preferentially expressed in human hematopoietic tissues, including CD34 + cells and leukemia cells. Its narrow tissue distribution, potential for substrate specificity, and close homology to other cell membrane transporters make OCT6 an attractive target for the treatment of leukemia.