The key nuclear export protein CRM1/XPO1 may represent a promising novel therapeutic target in human multiple myeloma (MM). Here we showed that chromosome region maintenance 1 (CRM1) is highly ...expressed in patients with MM, plasma cell leukemia cells and increased in patient cells resistant to bortezomib treatment. CRM1 expression also correlates with increased lytic bone and shorter survival. Importantly, CRM1 knockdown inhibits MM cell viability. Novel, oral, irreversible selective inhibitors of nuclear export (SINEs) targeting CRM1 (KPT-185, KPT-330) induce cytotoxicity against MM cells (ED50<200 nM), alone and cocultured with bone marrow stromal cells (BMSCs) or osteoclasts (OC). SINEs trigger nuclear accumulation of multiple CRM1 cargo tumor suppressor proteins followed by growth arrest and apoptosis in MM cells. They further block c-myc, Mcl-1, and nuclear factor κB (NF-κB) activity. SINEs induce proteasome-dependent CRM1 protein degradation; concurrently, they upregulate CRM1, p53-targeted, apoptosis-related, anti-inflammatory and stress-related gene transcripts in MM cells. In SCID mice with diffuse human MM bone lesions, SINEs show strong anti-MM activity, inhibit MM-induced bone lysis and prolong survival. Moreover, SINEs directly impair osteoclastogenesis and bone resorption via blockade of RANKL-induced NF-κB and NFATc1, with minimal impact on osteoblasts and BMSCs. These results support clinical development of SINE CRM1 antagonists to improve patient outcome in MM.
Drug resistance is a growing concern with clinical use of tyrosine kinase inhibitors. Utilizing in vitro models of intrinsic drug resistance and stromal-mediated chemoresistance, as well as ...functional mouse models of progressive and residual disease, we attempted to develop a potential therapeutic approach designed to suppress leukemia recurrence following treatment with selective kinase inhibitors. The novel IAP inhibitor, LCL161, corrected was observed to potentiate the effects of tyrosine kinase inhibition against leukemic disease both in the absence and presence of a stromal-protected corrected environment. LCL161 enhanced the proapoptotic effects of nilotinib and PKC412, against leukemic disease in vitro and potentiated the activity of both kinase inhibitors against leukemic disease in vivo. In addition, LCL161 synergized in vivo with nilotinib to reduce leukemia burden significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib. Finally, LCL161 displayed antiproliferative effects against cells characterized by intrinsic resistance to tyrosine kinase inhibitors as a result of expression of point mutations in the protein targets of drug inhibition. These results support the idea of using IAP inhibitors in conjunction with targeted tyrosine kinase inhibition to override drug resistance and suppress or eradicate residual disease.
Drugs that target the chief mediator of nuclear export, chromosome region maintenance 1 protein (CRM1) have potential as therapeutics for leukemia, but existing CRM1 inhibitors show variable ...potencies and a broad range of cytotoxic effects. Here, we report the structural analysis and antileukemic activity of a new generation of small-molecule inhibitors of CRM1. Designated selective inhibitors of nuclear export (SINE), these compounds were developed using molecular modeling to screen a small virtual library of compounds against the nuclear export signal (NES) groove of CRM1. The 2.2-Å crystal structure of the CRM1-Ran-RanBP1 complex bound to KPT-251, a representative molecule of this class of inhibitors, shows that the drug occupies part of the groove in CRM1 that is usually occupied by the NES, but penetrates much deeper into the groove and blocks CRM1-directed protein export. SINE inhibitors exhibit potent antileukemic activity, inducing apoptosis at nanomolar concentrations in a panel of 14 human acute myeloid leukemia (AML) cell lines representing different molecular subtypes of the disease. When administered orally to immunodeficient mice engrafted with human AML cells, KPT-251 had potent antileukemic activity with negligible toxicity to normal hematopoietic cells. Thus, KPT-SINE CRM1 antagonists represent a novel class of drugs that warrant further testing in AML patients.
Drug resistance is a growing area of concern. It has been shown that a small, residual pool of leukemic CD34+ progenitor cells can survive in the marrow microenvironment of chronic myeloid leukemia ...(CML) patients after years of kinase inhibitor treatment. Bone marrow (BM) stroma has been implicated in the long-term survival of leukemic cells, and contributes to the expansion and proliferation of both transformed and normal hematopoietic cells. Mechanistically, we found that CML cells expressed CXCR4, and that plerixafor diminished BCR-ABL-positive cell migration and reduced adhesion of these cells to extra cellular-matrix components and to BM stromal cells in vitro. Moreover, plerixafor decreased the drug resistance of CML cells induced by co-culture with BM stromal cells in vitro. Using a functional mouse model of progressive and residual disease, we demonstrated the ability of the CXCR4 inhibitor, plerixafor, to mobilize leukemic cells in vivo, such that a plerixafor-nilotinib combination reduced the leukemia burden in mice significantly below the baseline level suppression exhibited by a moderate-to-high dose of nilotinib as single agent. These results support the idea of using CXCR4 inhibition in conjunction with targeted tyrosine kinase inhibition to override drug resistance in CML and suppress or eradicate residual disease.
Mixed lineage leukemia (MLL)-fusion proteins can induce acute myeloid leukemias (AMLs) from either hematopoietic stem cells (HSCs) or granulocyte-macrophage progenitors (GMPs), but it remains unclear ...whether the cell of origin influences the biology of the resultant leukemia. MLL-AF9-transduced single HSCs or GMPs could be continuously replated, but HSC-derived clones were more likely than GMP-derived clones to initiate AML in mice. Leukemia stem cells derived from either HSCs or GMPs had a similar immunophenotype consistent with a maturing myeloid cell (LGMP). Gene expression analyses demonstrated that LGMP inherited gene expression programs from the cell of origin including high-level Evi-1 expression in HSC-derived LGMP. The gene expression signature of LGMP derived from HSCs was enriched in poor prognosis human MLL-rearranged AML in three independent data sets. Moreover, global 5'-mC levels were elevated in HSC-derived leukemias as compared with GMP-derived leukemias. This mirrored a difference seen in 5'-mC between MLL-rearranged human leukemias that are either EVI1 positive or EVI1 negative. Finally, HSC-derived leukemias were more resistant to chemotherapy than GMP-derived leukemias. These data demonstrate that the cell of origin influences the gene expression profile, the epigenetic state and the drug response in AML, and that these differences can account for clinical heterogeneity within a molecularly defined group of leukemias.
Acute myeloid leukemia (AML) progenitors are frequently characterized by activating mutations in the receptor tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). Protein tyrosine kinases are integral ...components of signaling cascades that have a role in both FLT3-mediated transformation as well as viability pathways that are advantageous to leukemic cell survival. The bone marrow microenvironment can diminish AML sensitivity to tyrosine kinase inhibitors. We hypothesized that inhibition of protein kinases in addition to FLT3 may be effective in overriding drug resistance in AML. We used a cell-based model mimicking stromal protection as part of an unbiased high-throughput chemical screen to identify kinase inhibitors with the potential to override microenvironment-mediated drug resistance in mutant FLT3-positive AML. Several related multi-targeted kinase inhibitors, including dasatinib, with the capability of reversing microenvironment-induced resistance to FLT3 inhibition were identified and validated. We validated synergy in vitro and demonstrated effective combination potential in vivo. In particular Janus kinase inhibitors were effective in overriding stromal protection and potentiating FLT3 inhibition in primary AML and cell lines. These results hint at a novel concept of using combination therapy to override drug resistance in mutant FLT3-positive AML in the bone marrow niche and suppress or eradicate residual disease.
Studies indicate that there is a positive relationship between openness to experience and creativity. However, relatively little attention has been given to the mechanism of this relationship. On the ...basis of previous findings, we hypothesized a conceptual model and tested the mediating role of intrinsic motivation and the creative process engagement in the relationship between openness to experience and creativity. One hundred and ninety‐eight undergraduates in Malaysia participated in the study and completed an online measure of openness to new experience, creativity, intrinsic motivation, and the creative process engagement. Consistent with the hypothesis, people who scored high on openness reported high intrinsic motivation. The high motivation enhanced engagement in creativity‐related activities, which in turn, improved self‐rated creativity. The findings not only shed light on mechanisms that underlie in the openness‐creativity linkage but they also highlight the importance of intrinsic motivation and creative process engagement in the linkage. Together, the study extends the effect of personality trait on creativity and offers a new direction for future studies.
Human leukemias harboring chromosomal translocations involving the mixed lineage leukemia (MLL, HRX, ALL-1) gene possess high-level expression, and frequent activating mutations of the receptor ...tyrosine kinase FLT3. We used a murine bone marrow transplant model to assess cooperation between MLL translocation and FLT3 activation. We demonstrate that MLL-AF9 expression induces acute myelogenous leukemia (AML) in approximately 70 days, whereas the combination of MLL-AF9 and FLT3-ITD does so in less than 30 days. Secondary transplantation of splenic cells from diseased mice established that leukemia stem cells are present at a very high frequency of approximately 1:100 in both diseases. Importantly, prospectively isolated granulocyte macrophage progenitors (GMPs) coinfected with MLL-AF9 and FLT3-ITD give rise to a similar AML, with shorter latency than from GMP transduced with MLL-AF9 alone. Cooperation between MLL-AF9 and FLT3-ITD was further verified by real-time assessment of leukemogenesis using noninvasive bioluminescence imaging. We used this model to demonstrate that MLL-AF9/FLT3-ITD-induced leukemias are sensitive to FLT3 inhibition in a 2-3 week in vivo assay. These data show that activated FLT3 cooperates with MLL-AF9 to accelerate onset of an AML from whole bone marrow as well as a committed hematopoietic progenitor, and provide a new genetically defined model system that should prove useful for rapid assessment of potential therapeutics in vivo.
The utility of cancer whole genome and transcriptome sequencing (cWGTS) in oncology is increasingly recognized. However, implementation of cWGTS is challenged by the need to deliver results within ...clinically relevant timeframes, concerns about assay sensitivity, reporting and prioritization of findings. In a prospective research study we develop a workflow that reports comprehensive cWGTS results in 9 days. Comparison of cWGTS to diagnostic panel assays demonstrates the potential of cWGTS to capture all clinically reported mutations with comparable sensitivity in a single workflow. Benchmarking identifies a minimum of 80× as optimal depth for clinical WGS sequencing. Integration of germline, somatic DNA and RNA-seq data enable data-driven variant prioritization and reporting, with oncogenic findings reported in 54% more patients than standard of care. These results establish key technical considerations for the implementation of cWGTS as an integrated test in clinical oncology.
We have developed a 3D dry lift-off process to localize multiple types of nitrifying bacteria in polyethylene glycol diacrylate (PEGDA) cubes for enhanced nitrification, a two-step biological process ...that converts ammonium to nitrite and then to nitrate. Ammonia-oxidizing bacteria (AOB) is responsible for converting ammonia into nitrite, and nitrite-oxidizing bacteria (NOB) is responsible for converting nitrite to nitrate. Successful nitrification is often challenging to accomplish, in part because AOB and NOB are slow growers and highly susceptible to many organic and inorganic chemicals in wastewater. Most importantly, the transportation of chemicals among scattered bacteria is extremely inefficient and can be problematic. For example, nitrite, produced from ammonia oxidation, is toxic to AOB and can lead to the failure of nitrification. To address these challenges, we closely localize AOB and NOB in PEGDA cubes as microenvironment modules to promote synergetic interactions. The AOB is first localized in the vicinity of the surface of the PEGDA cubes that enable AOB to efficiently uptake ammonia from a liquid medium and convert it into nitrite. The produced nitrite is then efficiently transported to the NOB localized at the center of the PEGDA particle and converted into non-toxic nitrate. Additionally, the nanoscale PEGDA fibrous structures offer a protective environment for these strains, defending them from sudden toxic chemical shocks and immobilize in cubes. This engineered microenvironment cube significantly enhances nitrification and improves the overall ammonia removal rate per single AOB cell. This approach-encapsulation of multiple strains at close range in cube in order to control their interactions-not only offers a new strategy for enhancing nitrification, but also can be adapted to improve the production of fermentation products and biofuel, because microbial processes require synergetic reactions among multiple species.
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