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Acute myeloid leukemia (AML) is an aggressive malignancy of stem cell origin that contributes to significant morbidity and mortality. The long-term prognosis remains dismal given the high likelihood for primary refractory or relapsed disease. An essential component of relapse is resurgence from the bone marrow. To date, the murine hematopoietic stem cell (HSC) niche has been clearly defined, but the human HSC niche is less well understood. The design of niche-based targeted therapies for AML must account for which cellular subsets compete for stem cell occupancy within respective bone marrow microenvironments. In this review, we highlight the principles of stem cell niche biology and discuss translational insights into the AML microenvironment as of 2021. Optimization of competition for niche occupancy is important for the elimination of measurable residual disease (MRD). Some of these novel therapeutics are in the pharmacologic pipeline for AML and may be especially useful in the setting of MRD.

Summary Most patients with AML succumb to chemoresistant leukemia stem cells (LSCs), which persist and reinitiate disease years after clinical remission. In this issue of Cell Stem Cell, Jones et al. (2020) identify a therapeutically targetable mechanism of resistance to venetoclax in relapsed and refractory AML LSCs mediated by nicotinamide metabolism.

Transient receptor protein (TRP) channels are frequently associated with tumors and are correlated to patient’s outcome. We firstly investigated TRP channel melastatin 8 (TRPM8) expression in urothelial carcinoma of bladder (UCB) and its correlation with UCB clinicopathological features and additionally evaluated the association between TRPM8 expression and patients’ survival rate to elucidate its role in bladder oncogenesis. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was conducted to examine TRPM8 messenger RNA (mRNA) expression in 36 pairs of freshly frozen UCB tissues and matched noncancerous tissues. Immunohistochemistry (IHC) was performed in 156 archived paraffin-embedded UCB samples to explore the correlation between TRPM8 protein and clinicopathological features. The association between TRPM8 expression and patient’s survival rate was evaluated using the Kaplan–Meier method and multivariate Cox regression analysis, respectively. The expression levels of TRPM8 mRNA in UCB tissues were significantly higher than those in matched noncancerous tissues (P = 0.016). Expression of TRPM8 protein in UCB was significantly and positively associated with histological grade (P = 0.039) and tumor stage (P = 0.037). Significant correlation between high TRPM8 expression and poor cumulative survival of UCB patients was shown using the Kaplan–Meier survival curve (P = 0.039). TRPM8 was represented as an independent prognostic biomarker for UCB patients by multivariate Cox regression analysis (P = 0.047). The present study provide the convincing evidence for the first time that over-expression of TRPM8 may play a role in the pathogenesis and progression of UCB, and TRPM8 may serve as an independent prognostic biomarker for UCB patients.

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34+38− cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34+38+ cells, SCF strongly induced pAKTS473 in a phosphoinositide 3-kinase (PI3K)–dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKTS473 levels remained low in CD34+38− cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34+38− compared with CD34+38+ CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells. Cancer Res; 73(18); 5775–86. ©2013 AACR.

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

11beta-Hydroxysteroid dehydrogenase type 1 (11betaHSD1) performs end-organ metabolism of glucocorticoids (GCs) by catalyzing the conversion of C(11)-keto-GCs to C(11)-hydroxy-GCs, thereby generating activating ligands for the GC receptor. In this study, we report that 11betaHSD1(-/-) mice are more susceptible to endotoxemia, evidenced by increased weight loss and serum TNF-alpha, IL-6, and IL-12p40 levels following LPS challenge in vivo. Peritoneal and splenic macrophage (splnMphi) from these genetically altered mice overproduce inflammatory cytokines following LPS stimulation in vitro. Inflammatory cytokine overexpression by 11betaHSD1(-/-) splnMphi results from an increased activation of NF-kappaB- and MAPK-signaling cascades and an attenuated PI3K-dependent Akt activation. The expression of SHIP1 is augmented in 11betaHSD1(-/-) Mphi and contributes to inflammatory cytokine production because overexpression of SHIP1 in primary bone marrow Mphi (BMMphi) leads to a similar type of hyperresponsiveness to subsequent LPS stimulation. 11betaHSD1(+/+) and 11betaHSD1(-/-) BMMphi responded to LPS similarly. However, 11betaHSD1(-/-) BMMphi derived in the presence of elevated GC levels up-regulated SHIP1 expression and increased their capacity to produce inflammatory cytokines following their activation with LPS. These observations suggest the hyperresponsiveness of 11betaHSD1(-/-) splnMphi results from myeloid cell differentiation in the presence of moderately elevated GC levels found within 11betaHSD1(-/-) mice. GC-conditioning of BMMphi enhanced SHIP1 expression via up-regulation of bioactive TGF-beta. Consistently, TGF-beta protein expression was increased in unstimulated CD11b(-) cells residing in the BM and spleen of 11betaHSD1(-/-) mice. Our results suggest that modest elevations in plasma GC levels can modify the LPS responsiveness of Mphi by augmenting SHIP1 expression through a TGF-beta-dependent mechanism.

Expression of the nuclear hormone receptor peroxisome proliferator-activated receptor α (PPARα) in resting lymphocytes was recently established, although the physiologic role(s) played by this nuclear hormone receptor in these cell types remains unresolved. In this study, we used CD4+ T cells isolated from PPARα−/− and wild-type mice, as well as cell lines that constitutively express PPARα, in experiments designed to evaluate the role of this hormone receptor in the regulation of T cell function. We report that activated CD4+ T cells lacking PPARα produce increased levels of IFN-γ, but significantly lower levels of IL-2 when compared with activated wild-type CD4+ T cells. Furthermore, we demonstrate that PPARα regulates the expression of these cytokines by CD4+ T cells in part, through its ability to negatively regulate the transcription of T-bet. The induction of T-bet expression in CD4+ T cells was determined to be positively influenced by p38 mitogen-activated protein (MAP) kinase activation, and the presence of unliganded PPARα effectively suppressed the phosphorylation of p38 MAP kinase. The activation of PPARα with highly specific ligands relaxed its capacity to suppress p38 MAP kinase phosphorylation and promoted T-bet expression. These results demonstrate a novel DNA-binding independent and agonist-controlled regulatory influence by the nuclear hormone receptor PPARα.

The immunomodulatory effects of glucocorticoids (GCs) have been described as bimodal, with high levels of GCs exerting immunosuppressive effects and low doses of GCs being immunopermissive. While the mechanisms used by GCs to achieve immunosuppression have been investigated intensely, the molecular mechanisms underlying the permissive effects of GCs remain uncharacterized. Herein, we demonstrate that GC conditioning during the differentiation of myeloid progenitors into macrophages (Mφs) results in their enhanced LPS responsiveness, demonstrated by an overexpression of the inflammatory cytokines TNF-α, IL-6, and IL-12. Inflammatory cytokine overexpression resulted from an increased activation of NF-κB and the MAPK signaling cascade and a reduced activation of the PI3K-Akt pathway following LPS stimulation. GC conditioning during Mφ differentiation induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway. Small interfering RNA-mediated knockdown of SHIP1 expression increased PI3K-dependent Akt activation and subsequently decreased inflammatory cytokine expression, suggesting GC-mediated up-regulation of SHIP1 expression is responsible for the augmentation in inflammatory cytokine production following LPS stimulation. We also show that splenic Mφs purified from normal mice that were implanted with timed-release GC pellets exhibited an enhanced LPS responsiveness and increased SHIP1 expression, indicating that GCs can regulate SHIP1 expression in vivo. Our results suggest that minor fluctuations in physiological levels of endogenous GCs can program endotoxin-responsive hemopoietic cells during their differentiation by regulating their sensitivity to stimulation.

Abstract 2778 In chronic myeloid leukemia (CML), imatinib and other tyrosine kinase inhibitors (TKIs) inhibit BCR-ABL1 tyrosine kinase activity but also target additional kinases including KIT. The role of KIT inhibition in the therapeutic efficacy of TKIs is controversial. We used TKIs with selective activity against ABL (PPY-A) or KIT (BAW667) and genetic tools to assess the role of KIT signaling for growth of CML cell lines and primary CML progenitor and stem cells. In Mo7eBCR-ABL1 or newly diagnosed CML CD34+ progenitor cells, immunoblotting confirmed that PPY-A (1 μM) suppresses BCR-ABL1 phosphorylation but not KIT tyrosine phosphorylation. In contrast, treatment of cells with a KIT-blocking antibody (K44.2, 200ng/mL), shRNA targeting KIT (shKIT), or the KIT selective inhibitor BAW667 (1 μM), suppressed KIT activity without affecting BCR-ABL1 kinase activity. Therefore, these systems are suitable to isolate the role of BCR-ABL1 vs. KIT inhibition. Treatment of Mo7eBCR-ABL1 cells with PPY-A resulted in suppression of growth by 91.7% (p To assess BCR-ABL1 vs. KIT inhibition in primary cells, CD34+ cells from newly diagnosed CML patients (n=4) and normal controls (n=3) were cultured in semisolid medium supplied with IL-3 and GM-CSF (no SCF), in the presence of 1 μM PPY-A combined with shKIT or 1 μM BAW667. KIT inhibition by shKIT or 1 μM BAW667 reduced CFU-GM formation by 40% compared to controls (p To assess the role of KIT vs. BCR-ABL1 inhibition on primitive CML cells we performed long-term culture-initiating cell (LTC-IC) assays on M2–10B4 murine stromal cells, using Lin− cells from newly diagnosed patients (n=3). Cultures were performed with K44.2, PPY-A, K44.2 plus PPY-A or 2 mM imatinib, with colonies plated at 1, 3, and 6 weeks. At 1 week colonies were reduced by 30% with K44.2 and 70% with PPY-A, but by 90% with the PPY-A / K44.2 combination or with imatinib. In contrast, at 6 weeks colony formation was unaffected by K44.2 but reduced by >95% with PPY-A, the PPY-A / K44.2 combination or imatinib. Week 3 colony growth was intermediate. Consistent with the LTC-IC assay, KIT inhibition with BAW667 enhanced PPY-A suppression of colony formation in Lin−CD34+CD38+ progenitor cells from newly diagnosed patients (n=3) by 18.7% (p Our findings suggest KIT inhibition is much more critical for suppression of mature progenitors compared to primitive CML cells. Since AKT is active in CML progenitors but suppressed by TGFβ in stem cells (Nature, 2010;463(7281):676; JCI, 2011;121(1):396), we speculate that upon BCR-ABL1 inhibition CML progenitors but not stem cells switch to an SCF-dependent mode of AKT activation, which renders these cells uniquely sensitive to dual inhibition of BCR-ABL1 and KIT signaling. Disclosures: No relevant conflicts of interest to declare.

Hepcidin is a peptide hormone that regulates iron homeostasis and acts as an antimicrobial peptide. It is expressed and secreted by a variety of cell types in response to iron loading and inflammation. Hepcidin mediates iron homeostasis by binding to the iron exporter ferroportin, inducing its internalization and degradation via activation of the protein kinase Jak2 and the subsequent phosphorylation of ferroportin. Here we have shown that hepcidin-activated Jak2 also phosphorylates the transcription factor Stat3, resulting in a transcriptional response. Hepcidin treatment of ferroportin-expressing mouse macrophages showed changes in mRNA expression levels of a wide variety of genes. The changes in transcript levels for half of these genes were a direct effect of hepcidin, as shown by cycloheximide insensitivity, and dependent on the presence of Stat3. Hepcidin-mediated transcriptional changes modulated LPS-induced transcription in both cultured macrophages and in vivo mouse models, as demonstrated by suppression of IL-6 and TNF-α transcript and secreted protein. Hepcidin-mediated transcription in mice also suppressed toxicity and morbidity due to single doses of LPS, poly(I:C), and turpentine, which is used to model chronic inflammatory disease. Most notably, we demonstrated that hepcidin pretreatment protected mice from a lethal dose of LPS and that hepcidin-knockout mice could be rescued from LPS toxicity by injection of hepcidin. The results of our study suggest a new function for hepcidin in modulating acute inflammatory responses.