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Acute myeloid leukemia (AML) kills 75% of patients and represents a major clinical challenge with a need to improve on current treatment approaches. Targeting sphingosine kinase 1 with a novel ATP-competitive-inhibitor, MP-A08, induces cell death in AML. However, limitations in MP-A08’s “drug-like properties” (solubility, biodistribution, and potency) hinder its pathway to the clinic. This study demonstrates a liposome-based delivery system of MP-A08 that exhibits enhanced MP-A08 potency against AML cells. MP-A08-liposomes increased MP-A08 efficacy against patient AML cells (>140-fold) and significantly prolonged overall survival of mice with human AML disease (P= 0.03). The significant antileukemic property of MP-A08-liposomes could be attributed to its enhanced specificity, bioaccessibility, and delivery to the bone marrow, as demonstrated in the pharmacokinetic and biodistribution studies. Our findings indicate that MP-A08-liposomes have potential as a novel treatment for AML.

Inducing cell death by the sphingolipid ceramide is a potential anticancer strategy, but the underlying mechanisms remain poorly defined. In this study, triggering an accumulation of ceramide in acute myeloid leukemia (AML) cells by inhibition of sphingosine kinase induced an apoptotic integrated stress response (ISR) through protein kinase R–mediated activation of the master transcription factor ATF4. This effect led to transcription of the BH3-only protein Noxa and degradation of the prosurvival Mcl-1 protein on which AML cells are highly dependent for survival. Targeting this novel ISR pathway, in combination with the Bcl-2 inhibitor venetoclax, synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anticancer effects of ceramide and preclinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

Inducing cell death by the sphingolipid ceramide is a potential anti-cancer strategy, but the underlying mechanisms remain poorly defined. Here, we show that triggering accumulation of ceramide in acute myeloid leukaemia (AML) cells by inhibition of sphingosine kinase induces an apoptotic integrated stress response (ISR) through protein kinase R-mediated activation of the master transcription factor ATF4. This leads to transcription of the BH3-only protein, Noxa, and degradation of the pro-survival Mcl-1 protein on which AML cells are highly dependent on for survival. Targeting this novel ISR pathway in combination with the Bcl-2 inhibitor venetoclax synergistically killed primary AML blasts, including those with venetoclax-resistant mutations, as well as immunophenotypic leukemic stem cells, and reduced leukemic engraftment in patient-derived AML xenografts. Collectively, these findings provide mechanistic insight into the anti-cancer effects of ceramide and pre-clinical evidence for new approaches to augment Bcl-2 inhibition in the therapy of AML and other cancers with high Mcl-1 dependency.

The tyrosine kinase receptor, EphB4, mediates cross‐talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin‐B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over‐expressing EphB4 under the control of collagen type‐1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long‐term culture‐initiating cells (LTC‐IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC‐IC in vitro, where blocking EphB4/ephrin‐B2 interactions decreased LTC‐IC output. Similarly, short hairpin RNA‐mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin‐B2 expressing CD34+HSC in LTC‐IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin‐1, IL‐6, FLT‐3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal‐mediated support of hematopoiesis, and constitute a novel component of the HSC niche. StemCells2015;33:2838—2849

In an era of pediatric emergency department (PED) overcrowding and diminishing health care resources, routine peripheral intravenous (PIV) catheter placement in the pediatric population requires evaluation because it might directly impact PED efficiency. This study aims to determine the utility of routine PIV catheter placement during phlebotomy.

High enantioselectivities are obtained for the reduction of a series of ketones using the inexpensive and mild reducing agent NaBH4 with TarB-NO2 (1). This easily prepared tartaric acid-based reagent combines a Lewis acid with carboxylic acids in a single bifunctional reagent. When combined with NaBH4, the resulting chiral acyloxyborohydride mediates the reduction of aromatic ketones to provide the product alcohols in enantiomeric excesses of 93–98 %. Several aliphatic ketones were also reduced with moderate to excellent enantioselectivity. A unique mechanism is provided with supporting calculations for the proposed active species and transition state. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

Oocyte-secreted growth differentiation factor 9 (GDF9) plays a critical role, in collaboration with FSH/EGF, during folliculogenesis through to ovulation. The molecular mechanism behind this dual requirement however remains unclear. One hypothesis on the relationship between these two pathways is that GDF9-activation of Smad2/3 enables FSH to exert its effects through MAPK signaling. Alternatively, this study investigated the need for MAPK3/1 signaling to allow oocyte/GDF9 to regulate key granulosa/cumulus cell functions. In the absence of FSH or EGF, 3H-thymidine incorporation and upregulation of matrix genes (Ptx3 and Has2) in granulosa cells stimulated by oocyte coculture or recombinant mouse GDF9 was, as expected, inhibited by SB431542 (5 µM), which prevents Smad2/3 activation. Surprisingly, this oocyte/GDF9 effect was inhibited as well by the EGF receptor inhibitor AG1478 (5 µM), despite the absence of FSH and EGF. This suggests that a minimal "basal" level of EGF receptor activity is required for oocyte/GDF9 bioactivity in granulosa cells. In addition to inhibition by SB431542 and AG1478, the oocyte- and GDF9-stimulated 3H-thymidine incorporation was inhibited by the MEK1 inhibitor U0126 (10 µM) and the Src inhibitor PP2 (10 µM), while the bone morphogenetic protein (BMP) type 1 receptor Alk2/3/6 inhibitor dorsomorphin (10 μM) was without effect. GDF9-stimulated 3H-thymidine incorporation was not affected by the NF-κB inhibitor SN50 (10 µg/ml) or the matrix metalloproteinase inhibitor GM6001 (10 µM), excluding the NF-κB pathway or shedding of membrane-bound EGF ligands as participating in GDF9 bioactivity. Interestingly, activin A (10 ng/ml)- and TGFβ1 (2 ng/ml)-stimulated 3H-thymidine incorporation had similar inhibitor sensitivity. When treated as cumulus-oocyte complexes, cumulus cell 3H-thymidine incorporation was also inhibited by the Alk4/5/7, EGF receptor and Src inhibitors. These results suggest that growth factors signaling through the Smad2/3 pathway not only need an active type 1 receptor, but also share a common requirement of EGF receptor-dependant and Src-dependant MAPK3/1 phosphorylation. Western blot analysis showed that granulosa cells have a basal (ligand-independant) level of MAPK3/1 phosphorylation that can be inhibited by using an EGF receptor or a Src inhibitor and increased by treating with EGF. EGF-stimulated MAPK3/1 phosphorylation was not affected by inhibition of Alk4/5/7 or by adding exogenous recombinant GDF9. Using a construct possessing a Smad3-activated promoter coupled with the luciferase gene transfected into mouse granulosa cells, GDF9-stimulated promoter activation was inhibited by Alk4/5/7, EGF receptor, MEK1 and Src inhibitors. Conversely, GDF9-stimulated Smad3-promoter activity was increased in a dose-dependent manner by EGF. These experiments suggest that MAPK3/1 activation amplifies Smad2/3-mediated growth factor effects on granulosa/cumulus cells, while MAPK3/1 inhibition abolishes their bioactivity. These results lead us to hypothesize that, Src/EGFR-dependant MAPK3/1 phosphorylation is required for oocyte/GDF9-stimulated functions of granulosa and cumulus cells. Hence, apart from its role in the transmission of the ovulatory LH signal, EGF-like molecules might serve as a critical modulators of GDF9 action on granulosa/cumulus cells. This research was supported by the NHMRC of Australia to RBG and DLR and a postdoctoral fellowship from NSERC of Canada to MS.(platform)

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