Summary Myelodysplastic Syndromes (MDS) arise from a defective hematopoietic stem/progenitor cell. Consequently, there is an urgent need to develop targeted therapies capable of eliminating the MDS-initiating clones. We identified that IRAK1, an immune modulating kinase, is overexpressed and hyperactivated in MDS. MDS clones treated with a small-molecule IRAK1 inhibitor (IRAK1/4-Inh) exhibited impaired expansion and increased apoptosis, which coincided with TRAF6/NF- κB inhibition. Suppression of IRAK1, either by RNAi or with IRAK1/4-Inh, is detrimental to MDS cells while sparing normal CD34+ cells. Based on an integrative gene expression analysis, we combined IRAK1 and BCL2 inhibitors and found that co-treatment more effectively eliminated MDS clones. In summary, these findings implicate IRAK1 as a drugable target in MDS.

IntroductionMyelodysplastic syndrome (MDS) is a hematologic malignancy defined by blood cytopenias due to ineffective hematopoiesis, a predisposition to acute myeloid leukemia (AML), and genomic instability. 1,2 Molecular-targeted therapies do not exist for MDS and the mechanisms of current therapies are largely unknown. More recently, bortezomib (Velcade), which is widely used for the treatment of multiple myeloma (MM) and lymphomas, is being evaluated as a single agent or in combination with chemotherapy in certain MDS and AML patients. [3][4][5] Bortezomib is a selective and reversible inhibitor of the 26S proteasome, and mechanistic studies have revealed that inhibition of the proteasome complex leads to accumulation of lysine (K)-48 ubiquitin-linked proteins and consequently to cytotoxic effects in malignant cells. 6 Proapoptotic and cell-cycle inhibitor proteins are stabilized after proteasome inhibition and thought to contribute to the anticancer effect by inducing apoptosis and inhibiting the cell cycle, respectively. 6 Nevertheless, the molecular and cellular mechanisms of bortezomib-induced cytotoxicity remain unknown, particularly in MDS/AML. Whereas the role of bortezomib in regulating cell-cycle entry and survival have been characterized partially in MDS/AML, 7-9 recent evidence has pointed to a more general cellular effect: bortezomib treatment results in the accumulation of nondegraded proteins, leading to endoplasmic reticulum stress and autophagy in cancer. [10][11][12] Under normal cellular stresses, autophagy, a catabolic pathway, degrades long-lived proteins and defective and superfluous organelles. 13 However, under conditions of extreme cellular stress, autophagy is used by the cell to undergo death. 14 Human miR-146a, a candidate gene in del(5q) MDS/AML, is reduced significantly in del(5q) and normal karyotype MDS/AML patients. [15][16][17] TRAF6 is a key target of miR-146a 15,18,19 and, as expected, miR-146a-knockout mice have a dramatic increase in TRAF6 protein within the hematopoietic compartment. 20,21 Retroviral overexpression of TRAF6 in mouse hematopoietic stem/ progenitor cells results in MDS-like hematopoietic defects and progression to AML. 15 Bortezomib has been shown previously to be effective for an MDS patient with del(5q) and was also reported to reduce directly TRAF6 mRNA and protein in osteoclast precursors from MM patients. 22,23 Because TRAF6 is implicated in MDS/AML and bortezomib has been shown to be effective in del(5q) MDS and to inhibit TRAF6 in MM, we hypothesized that one mechanism of bortezomib action is through inhibition of TRAF6. The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 12, 2018. by guest www.bloodjournal.org FromIn the present study, we identified TRAF6 as a relevant target of bortezomib-induced...

Proinflammatory cytokines have an important pathophysiologic role in septic shock. CD14 is involved in cytokine responses to a number of purified bacterial products, including LPS. However, little is known of monocyte receptors involved in cytokine responses to whole bacteria. To identify these receptors, human monocytes were pretreated with different mAbs and TNF-α was measured in culture supernatants after stimulation with whole heat-killed bacteria. Human serum and anti-CD14 Abs significantly increased and decreased, respectively, TNF-α responses to the Gram-negative Escherichia coli. However, neither treatment influenced responses to any of the Gram-positive bacteria tested, including group A and B streptococci, Listeria monocytogenes, and Staphylococcus aureus. Complement receptor type III (CR3 or CD18/CD11b) Abs prevented TNF-α release induced by heat-killed group A or B streptococci. In contrast, the same Abs had no effects when monocytes were stimulated with L. monocytogenes or S. aureus. Using either of the latter bacteria, significant inhibition of TNF-α release was produced by Abs to CD11c, one of the subunits of CR4. To confirm these blocking Ab data, IL-6 release was measured in CR3-, CR4-, or CD14-transfected Chinese hamster ovary cells after bacterial stimulation. Accordingly, streptococci triggered moderate IL-6 production (p < 0.05) in CR3 but not CD14 or CR4 transfectants. In contrast, L. monocytogenes and S. aureus induced IL-6 release in CR4 but not CR3 or CD14 transfectants. Collectively our data indicate that β2 integrins, such as CR3 and CR4, may be involved in cytokine responses to Gram-positive bacteria. Moreover, CD14 may play a more important role in responses to whole Gram-negative bacteria relative to Gram-positive ones.

Despite the high response rates of individuals with myelodysplastic syndrome (MDS) with deletion of chromosome 5q (del(5q)) to treatment with lenalidomide (LEN) and the recent identification of cereblon (CRBN) as the molecular target of LEN, the cellular mechanism by which LEN eliminates MDS clones remains elusive. Here we performed an RNA interference screen to delineate gene regulatory networks that mediate LEN responsiveness in an MDS cell line, MDSL. We identified GPR68, which encodes a G-protein-coupled receptor that has been implicated in calcium metabolism, as the top candidate gene for modulating sensitivity to LEN. LEN induced GPR68 expression via IKAROS family zinc finger 1 (IKZF1), resulting in increased cytosolic calcium levels and activation of a calcium-dependent calpain, CAPN1, which were requisite steps for induction of apoptosis in MDS cells and in acute myeloid leukemia (AML) cells. In contrast, deletion of GPR68 or inhibition of calcium and calpain activation suppressed LEN-induced cytotoxicity. Moreover, expression of calpastatin (CAST), an endogenous CAPN1 inhibitor that is encoded by a gene (CAST) deleted in del(5q) MDS, correlated with LEN responsiveness in patients with del(5q) MDS. Depletion of CAST restored responsiveness of LEN-resistant non-del(5q) MDS cells and AML cells, providing an explanation for the superior responses of patients with del(5q) MDS to LEN treatment. Our study describes a cellular mechanism by which LEN, acting through CRBN and IKZF1, has cytotoxic effects in MDS and AML that depend on a calcium- and calpain-dependent pathway.

Type 2 diabetes mellitus (T2DM) is associated with endothelial dysfunction, which leads to vascular complications. Endothelial progenitor cells (EPCs) are thought to be a subset of cells derived from the bone marrow that play a crucial role in the neovascularization of ischemic tissue and in the maintenance of endothelial cell integrity. In contrast, circulating endothelial cells (CECs) are of endothelial origin and become detached from the intima of blood vessels in response to pathological stimuli. The study investigated the effects of T2DM on subpopulations of EPCs and CECs in peripheral blood, as compared with the effects on unacylated (UAG) and acylated (AG) ghrelin levels, which have been shown recently to play an important role in endothelial dysfunction associated with diabetes. Using the high-performance flow cytometer FACSCanto, and UAG/AG ghrelin enzyme immunoassay kits, we analyzed whole peripheral blood samples from: (i) diabetic patients with a history of disease of less than 1 year and no clinical evidence of angiopathy, (ii) diabetic patients with long-standing disease with vascular complications, and (iii) healthy donors. We found that T2DM did not affect bone-marrow mobilization, but it altered the UAG/AG profile and decreased the number of highly differentiated EPCs (late EPCs) greatly. In addition, T2DM increased the number of CECs, together with the number of activated CECs. Our results suggest that: (i) the endothelial damage could be due mainly to altered maturation/commitment of EPCs, rather than a simple decrease in their production in the bone marrow; and (ii) EPC subpopulations and ghrelin levels could be useful markers to assess endothelial damage in diabetes. ' 2012 International Society for Advancement of Cytometry