An Indole-Chalcone Inhibits Multidrug-Resistant Cancer Cell Growth by Targeting Microtubules.

Multidrug resistance and toxic side effects are the major challenges in cancer treatment with microtubule-targeting agents (MTAs), and thus, there is an urgent clinical need for new therapies. Chalcone, a common simple scaffold found in many natural products, is widely used as a privileged structure in medicinal chemistry. We have previously validated tubulin as the anticancer target for chalcone derivatives. In this study, an α-methyl-substituted indole-chalcone (FC77) was synthesized and found to exhibit an excellent cytotoxicity against the NCI-60 cell lines (average concentration causing 50% growth inhibition = 6 nM). More importantly, several multidrug-resistant cancer cell lines showed no resistance to FC77, and the compound demonstrated good selective toxicity against cancer cells versus normal CD34+ blood progenitor cells. A further mechanistic study demonstrated that FC77 could arrest cells that relate to the binding to tubulin and inhibit the microtubule dynamics. The National Cancer Institute COMPARE analysis and molecular modeling indicated that FC77 had a mechanism of action similar to that of colchicine. Overall, our data demonstrate that this indole-chalcone represents a novel MTA template for further development of potential drug candidates for the treatment of multidrug-resistant cancers.

Stat5 is critical for the development and maintenance of myeloproliferative neoplasm initiated by Nf1 deficiency.

Juvenile myelomonocytic leukemia is a rare myeloproliferative neoplasm characterized by hyperactive RAS signaling. Neurofibromin1 (encoded by the NF1 gene) is a negative regulator of RAS activation. Patients with neurofibromatosis type 1 harbor loss-of-function mutations in NF1 and have a 200- to 500-fold increased risk of juvenile myelomonocytic leukemia. Leukemia cells from patients with juvenile myelomonocytic leukemia display hypersensitivity to certain cytokines, such as granulocyte-macrophage colony-stimulating factor. The granulocyte-macrophage colony-stimulating factor receptor utilizes pre-associated JAK2 to initiate signals after ligand binding. JAK2 subsequently activates STAT5, among other downstream effectors. Although STAT5 is gaining recognition as an important mediator of growth factor signaling in myeloid leukemias, the contribution of STAT5 to the development of hyperactive RAS-initiated myeloproliferative disease has not been well described. In this study, we investigated the consequence of STAT5 attenuation via genetic and pharmacological approaches in Nf1-deficient murine models of juvenile myelomonocytic leukemia. We found that homozygous Stat5 deficiency extended the lifespan of Nf1-deficient mice and eliminated the development of myeloproliferative neoplasm associated with Nf1 gene loss. Likewise, we found that JAK inhibition with ruxolitinib attenuated myeloproliferative neoplasm in Nf1-deficient mice. Finally, we found that primary cells from a patient with KRAS-mutant juvenile myelomonocytic leukemia displayed reduced colony formation in response to JAK2 inhibition. Our findings establish a central role for STAT5 activation in the pathogenesis of juvenile myelomonocytic leukemia and suggest that targeting this pathway may be of clinical utility in these patients.

Targeting Ras signaling in AML: RALB is a small GTPase with big potential.

Acute myeloid leukemia (AML) is a devastating malignancy for which novel treatment approaches are desperately needed. Ras signaling is an attractive therapeutic target for AML because a large proportion of AMLs have mutations in NRAS, KRAS, or genes that activate Ras signaling, and key Ras effectors are activated in virtually all AML patient samples. This has inspired efforts to develop Ras-targeted treatment strategies for AML. Due to the inherent difficulty and disappointing efficacy of targeting Ras proteins directly, many have focused on inhibiting Ras effector pathways. Inhibiting the major oncogenic Ras effectors, the mitogen-activated protein kinase (MAPK) and/or phosphatidylinositiol-3-kinase (PI3K) pathways, has generally demonstrated modest efficacy for AML. While this may be in part related to functional redundancy between these pathways, it is now clear that other Ras effectors have key oncogenic roles. Specifically, the Ras-like (Ral) GTPases have emerged as critical mediators of Ras-driven transformation and AML cell survival. Our group recently uncovered a critical role for RALB signaling in leukemic cell survival and a potential mediator of relapse following Ras-targeted therapy in AML. Furthermore, we found that RALB signaling is hyperactivated in AML patient samples, and inhibiting RALB has potent anti-leukemic activity in preclinical AML models. While key questions remain regarding the importance of RALB signaling across the genetically diverse spectrum of AML, the specific mechanism(s) that promotes leukemic cell survival downstream of RALB, and how to pharmacologically target RALB signaling effectively - RALB has emerged as a critical Ras effector and potential therapeutic target for AML.

Potent Cytolytic Activity and Specific IL15 Delivery in a Second-Generation Trispecific Killer Engager.

Natural killer (NK) cells are potent immune modulators that can quickly lyse tumor cells and elicit inflammatory responses. These characteristics make them ideal candidates for immunotherapy. However, unlike T cells, NK cells do not possess clonotypic receptors capable of specific antigen recognition and cannot expand via activating receptor signals alone. To enable NK cells with these capabilities, we created and have previously described a tri-specific killer engager (TriKE) platform capable of inducing antigen specificity and cytokine-mediated NK-cell expansion. TriKE molecules have three arms: (i) a single-chain variable fragment (scFv) against the activating receptor CD16 on NK cells to trigger NK-cell activation, (ii) an scFv against a tumor-associated antigen (CD33 here) to induce specific tumor target recognition, and (iii) an IL15 moiety to trigger NK-cell expansion and priming. Here, we demonstrate that by modifying the anti-CD16 scFv with a humanized single-domain antibody against CD16, we improved TriKE functionality. A CD33-targeting second-generation TriKE induced stronger and more specific NK-cell proliferation without T-cell stimulation, enhanced in vitro NK-cell activation and killing of CD33-expressing targets, and improved tumor control in preclinical mouse models. Given these improved functional characteristics, we propose rapid translation of second-generation TriKEs into the clinic.

Functional analysis of human hematopoietic stem cell gene expression using zebrafish.

Although several reports have characterized the hematopoietic stem cell (HSC) transcriptome, the roles of HSC-specific genes in hematopoiesis remain elusive. To identify candidate regulators of HSC fate decisions, we compared the transcriptome of human umbilical cord blood and bone marrow (CD34+)(CD33-)(CD38-)Rho(lo)(c-kit+) cells, enriched for hematopoietic stem/progenitor cells with (CD34+)(CD33-)(CD38-)Rho(hi) cells, enriched in committed progenitors. We identified 277 differentially expressed transcripts conserved in these ontogenically distinct cell sources. We next performed a morpholino antisense oligonucleotide (MO)-based functional screen in zebrafish to determine the hematopoietic function of 61 genes that had no previously known function in HSC biology and for which a likely zebrafish ortholog could be identified. MO knock down of 14/61 (23%) of the differentially expressed transcripts resulted in hematopoietic defects in developing zebrafish embryos, as demonstrated by altered levels of circulating blood cells at 30 and 48 h postfertilization and subsequently confirmed by quantitative RT-PCR for erythroid-specific hbae1 and myeloid-specific lcp1 transcripts. Recapitulating the knockdown phenotype using a second MO of independent sequence, absence of the phenotype using a mismatched MO sequence, and rescue of the phenotype by cDNA-based overexpression of the targeted transcript for zebrafish spry4 confirmed the specificity of MO targeting in this system. Further characterization of the spry4-deficient zebrafish embryos demonstrated that hematopoietic defects were not due to more widespread defects in the mesodermal development, and therefore represented primary defects in HSC specification, proliferation, and/or differentiation. Overall, this high-throughput screen for the functional validation of differentially expressed genes using a zebrafish model of hematopoiesis represents a major step toward obtaining meaningful information from global gene profiling of HSCs.

Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

RALB provides critical survival signals downstream of Ras in acute myeloid leukemia.

Mutations that activate RAS proto-oncogenes and their effectors are common in acute myeloid leukemia (AML); however, efforts to therapeutically target Ras or its effectors have been unsuccessful, and have been hampered by an incomplete understanding of which effectors are required for AML proliferation and survival. We investigated the role of Ras effector pathways in AML using murine and human AML models. Whereas genetic disruption of NRAS(V12) expression in an NRAS(V12) and Mll-AF9-driven murine AML induced apoptosis of leukemic cells, inhibition of phosphatidylinositol-3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) signaling did not reproduce this effect. Conversely, genetic disruption of RALB signaling induced AML cell death and phenocopied the effects of suppressing oncogenic Ras directly - uncovering a novel role for RALB signaling in AML survival. Knockdown of RALB led to decreased phosphorylation of TBK1 and reduced BCL2 expression, providing mechanistic insight into RALB survival signaling in AML. Notably, we found that patient-derived AML blasts have higher levels of RALB-TBK1 signaling compared to normal blood leukocytes, supporting a pathophysiologic role for RALB signaling for AML patients. Overall, our work provides new insight into the specific roles of Ras effector pathways in AML and has identified RALB signaling as a key survival pathway.

The transcription factors Scl and Lmo2 act together during development of the hemangioblast in zebrafish.

The transcription factors Scl and Lmo2 are crucial for development of all blood. An important early requirement for Scl in endothelial development has also been revealed recently in zebrafish embryos, supporting previous findings in scl(-/-) embryoid bodies. Scl depletion culminates most notably in failure of dorsal aorta formation, potentially revealing a role in the formation of hemogenic endothelium. We now present evidence that the requirements for Lmo2 in zebrafish embryos are essentially the same as for Scl. The expression of important hematopoietic regulators is lost, reduced, or delayed, panendothelial gene expression is down-regulated, and aorta-specific marker expression is lost. The close similarity of the phenotypes for Scl and Lmo2 suggest that they perform these early functions in hemangioblast development within a multiprotein complex, as shown for erythropoiesis. Consistent with this, we find that scl morphants cannot be rescued by a non-Lmo2-binding form of Scl but can be rescued by non-DNA-binding forms, suggesting tethering to target genes through DNA-binding partners linked via Lmo2. Interestingly, unlike other hematopoietic regulators, the Scl/Lmo2 complex does not appear to autoregulate, as neither gene's expression is affected by depletion of the other. Thus, expression of these critical regulators is dependent on continued expression of upstream regulators, which may include cell-extrinsic signals.

Genome-wide reverse genetics framework to identify novel functions of the vertebrate secretome.

BACKGROUND: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. RESULTS: We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies. CONCLUSION: This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.

The molecular repertoire of the 'almighty' stem cell.

Stem cells share the defining characteristics of self-renewal, which maintains or expands the stem-cell pool, and multi-lineage differentiation, which generates and regenerates tissues. Stem-cell self-renewal and differentiation are influenced by the convergence of intrinsic cellular signals and extrinsic microenvironmental cues from the surrounding stem-cell niche, but the specific signals involved are poorly understood. Recently, several studies have sought to identify the genetic mechanisms that underlie the stem-cell phenotype. Such a molecular road map of stem-cell function should lead to an understanding of the true potential of stem cells.