Genomic profiling and directed ex vivo drug analysis of an unclassifiable myelodysplastic/myeloproliferative neoplasm progressing into acute myeloid leukemia.

Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs. © 2016 Wiley Periodicals, Inc.

SMS121, a new inhibitor of CD36, impairs fatty acid uptake and viability of acute myeloid leukemia.

Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common among children. AML is characterized by aberrant proliferation of myeloid blasts in the bone marrow and impaired normal hematopoiesis. Despite the introduction of new drugs and allogeneic bone marrow transplantation, patients have poor overall survival rate with relapse as the major challenge, driving the demand for new therapeutic strategies. AML patients with high expression of the very long/long chain fatty acid transporter CD36 have poorer survival and very long chain fatty acid metabolism is critical for AML cell survival. Here we show that fatty acids are transferred from human primary adipocytes to AML cells upon co-culturing. A drug-like small molecule (SMS121) was identified by receptor-based virtual screening and experimentally demonstrated to target the lipid uptake protein CD36. SMS121 reduced the uptake of fatty acid into AML cells that could be reversed by addition of free fatty acids and caused decreased cell viability. The data presented here serves as a framework for the development of CD36 inhibitors to be used as future therapeutics against AML.

Sequential drug treatment targeting cell cycle and cell fate regulatory programs blocks non-genetic cancer evolution in acute lymphoblastic leukemia.

BACKGROUND: Targeted therapies exploiting vulnerabilities of cancer cells hold promise for improving patient outcome and reducing side-effects of chemotherapy. However, efficacy of precision therapies is limited in part because of tumor cell heterogeneity. A better mechanistic understanding of how drug effect is linked to cancer cell state diversity is crucial for identifying effective combination therapies that can prevent disease recurrence. RESULTS: Here, we characterize the effect of G2/M checkpoint inhibition in acute lymphoblastic leukemia (ALL) and demonstrate that WEE1 targeted therapy impinges on cell fate decision regulatory circuits. We find the highest inhibition of recovery of proliferation in ALL cells with KMT2A-rearrangements. Single-cell RNA-seq and ATAC-seq of RS4;11 cells harboring KMT2A::AFF1, treated with the WEE1 inhibitor AZD1775, reveal diversification of cell states, with a fraction of cells exhibiting strong activation of p53-driven processes linked to apoptosis and senescence, and disruption of a core KMT2A-RUNX1-MYC regulatory network. In this cell state diversification induced by WEE1 inhibition, a subpopulation transitions to a drug tolerant cell state characterized by activation of transcription factors regulating pre-B cell fate, lipid metabolism, and pre-BCR signaling in a reversible manner. Sequential treatment with BCR-signaling inhibitors dasatinib, ibrutinib, or perturbing metabolism by fatostatin or AZD2014 effectively counteracts drug tolerance by inducing cell death and repressing stemness markers. CONCLUSIONS: Collectively, our findings provide new insights into the tight connectivity of gene regulatory programs associated with cell cycle and cell fate regulation, and a rationale for sequential administration of WEE1 inhibitors with low toxicity inhibitors of pre-BCR signaling or metabolism.

Combined GLUT1 and OXPHOS inhibition eliminates acute myeloid leukemia cells by restraining their metabolic plasticity.

Acute myeloid leukemia (AML) is initiated and propagated by leukemia stem cells (LSCs), a self-renewing population of leukemia cells responsible for therapy resistance. Hence, there is an urgent need to identify new therapeutic opportunities targeting LSCs. Here, we performed an in vivo CRISPR knockout screen to identify potential therapeutic targets by interrogating cell surface dependencies of LSCs. The facilitated glucose transporter type 1 (GLUT1) emerged as a critical in vivo metabolic dependency for LSCs in a murine MLL::AF9-driven model of AML. GLUT1 disruption by genetic ablation or pharmacological inhibition led to suppression of leukemia progression and improved survival of mice that received transplantation with LSCs. Metabolic profiling revealed that Glut1 inhibition suppressed glycolysis, decreased levels of tricarboxylic acid cycle intermediates and increased the levels of amino acids. This metabolic reprogramming was accompanied by an increase in autophagic activity and apoptosis. Moreover, Glut1 disruption caused transcriptional, morphological, and immunophenotypic changes, consistent with differentiation of AML cells. Notably, dual inhibition of GLUT1 and oxidative phosphorylation (OXPHOS) exhibited synergistic antileukemic effects in the majority of tested primary AML patient samples through restraining of their metabolic plasticity. In particular, RUNX1-mutated primary leukemia cells displayed striking sensitivity to the combination treatment compared with normal CD34+ bone marrow and cord blood cells. Collectively, our study reveals a GLUT1 dependency of murine LSCs in the bone marrow microenvironment and demonstrates that dual inhibition of GLUT1 and OXPHOS is a promising therapeutic approach for AML.

Blockade of NKG2D ameliorates disease in mice with collagen-induced arthritis: a potential pathogenic role in chronic inflammatory arthritis.

OBJECTIVE: To assess the role of the activating receptor NKG2D in arthritis. METHODS: Levels of NKG2D and its ligands were determined by fluorescence-activated cell sorting, real-time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti-NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days. RESULTS: We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae-1 was up-regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down-modulated during the progression of collagen-induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti-NKG2D-treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti-NKG2D treatment significantly reduced both interleukin-17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro. CONCLUSION: Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

Duplex Sequencing Uncovers Recurrent Low-frequency Cancer-associated Mutations in Infant and Childhood KMT2A-rearranged Acute Leukemia.

Infant acute lymphoblastic leukemia (ALL) with KMT2A-gene rearrangements (KMT2A-r) have few mutations and a poor prognosis. To uncover mutations that are below the detection of standard next-generation sequencing (NGS), a combination of targeted duplex sequencing and NGS was applied on 20 infants and 7 children with KMT2A-r ALL, 5 longitudinal and 6 paired relapse samples. Of identified nonsynonymous mutations, 87 had been previously implicated in cancer and targeted genes recurrently altered in KMT2A-r leukemia and included mutations in KRAS, NRAS, FLT3, TP53, PIK3CA, PAX5, PIK3R1, and PTPN11, with infants having fewer such mutations. Of identified cancer-associated mutations, 62% were below the resolution of standard NGS. Only 33 of 87 mutations exceeded 2% of cellular prevalence and most-targeted PI3K/RAS genes (31/33) and typically KRAS/NRAS. Five patients only had low-frequency PI3K/RAS mutations without a higher-frequency signaling mutation. Further, drug-resistant clones with FLT3 D835H or NRAS G13D/G12S mutations that comprised only 0.06% to 0.34% of diagnostic cells, expanded at relapse. Finally, in longitudinal samples, the relapse clone persisted as a minor subclone from diagnosis and through treatment before expanding during the last month of disease. Together, we demonstrate that infant and childhood KMT2A-r ALL harbor low-frequency cancer-associated mutations, implying a vast subclonal genetic landscape.

Lactate contributes to ammonia-mediated astroglial dysfunction during hyperammonemia.

Even though ammonia is considered to underlie nervous system symptoms of dysfunction during hyperammonemia, lactate, which increases as a metabolic consequence of high ammonia levels, might also be a contributing factor. The data presented here show that NH4Cl (5 mM) mediates astroglial cell swelling, and that treatment with NH4Cl or lactate (25 mM) causes rearrangements of actin filaments and reduces astroglial glutamate uptake capacity. Co-application with BaCl2, which blocks astroglial uptake of NH4+, prevents NH4Cl-mediated cell swelling and rearrangement of actin filaments, but does not reduce NH4Cl-induced glutamate uptake capacity inhibition. Neither NH4Cl nor lactate affected glutamate uptake or protein expression in microglial cultures, indicating that astroglial cells are more susceptible to the neurotoxic affects of ammonia. Our results suggest that ammonium underlies brain edema, but that lactate can contribute to some of the cellular dysfunctions associated with elevated cerebral levels of ammonia.

The Conundrum of GSK3 Inhibitors: Is it the Dawn of a New Beginning?

Spanning over three decades of extensive drug discovery research, the efforts to develop a potent and selective GSK3 inhibitor as a therapeutic for the treatment of type 2 diabetes, Alzheimer's disease (AD), bipolar disorders and cancer have been futile. Since its initial discovery in 1980 and subsequent decades of research, one cannot underscore the importance of the target and the promise of a game changing disease modifier. Several pharmaceutical companies, biotech companies, and academic institutions raged in a quest to unravel the biology and discover potent and selective GSK3 inhibitors, some of which went through clinical trials. However, the conundrum of what happened to the fate of the AstraZeneca's GSK3 inhibitors and the undertaking to find a therapeutic that could control glycogen metabolism and aberrant tau hyperphosphorylation in the brain, and rescue synaptic dysfunction has largely been untold. AstraZeneca was in the forefront of GSK3 drug discovery research with six GSK3 drug candidates, one of which progressed up to Phase II clinical trials in the quest to untangle the tau hypothesis for AD. Analysis of key toxicity issues, serendipitous findings and efficacy, and biomarker considerations in relation to safety margins have limited the potential of small molecule therapeutics as a way forward. To guide future innovation of this important target, we reveal the roller coaster journey comprising of two decades of preclinical and clinical GSK3 drug discovery at AstraZeneca; the understanding of which could lead to improved GSK3 therapies for disease. These learnings in combination with advances in achieving kinase selectivity, different modes of action as well as the recent discovery of novel conjugated peptide technology targeting specific tissues have potentially provided a venue for scientific innovation and a new beginning for GSK3 drug discovery.

De novo activating mutations drive clonal evolution and enhance clonal fitness in KMT2A-rearranged leukemia.

Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3 ITD , FLT3 N676K , and NRAS G12D accelerate KMT2A-MLLT3 leukemia onset. Further, also subclonal FLT3 N676K mutations accelerate disease, possibly by providing stimulatory factors. Herein, we show that one such factor, MIF, promotes survival of mouse KMT2A-MLLT3 leukemia initiating cells. We identify acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 in KMT2A-MLLT3 leukemia cells that favored clonal expansion. During clonal evolution, we observe serial genetic changes at the Kras G12D locus, consistent with a strong selective advantage of additional Kras G12D . KMT2A-MLLT3 leukemias with signaling mutations enforce Myc and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlight the importance of activated signaling as a contributing driver.

Alteration of the cortisol-cortisone shuttle in leprosy type 1 reactions in leprosy patients in Hyderabad, India.

Regulation of inflammation in leprosy may be influenced by local concentrations of active cortisol and inactive cortisone, whose concentrations are regulated by enzymes in the cortisol-cortisone shuttle. We investigated the cortisol-cortisone shuttle enzymes in the skin of leprosy patients with type 1 reactions (T1R), which are characterised by skin and nerve inflammation. Gene expression of the shuttle enzymes were quantified in skin biopsies from 15 leprosy patients with new T1R before and during prednisolone treatment and compared with levels in skin biopsies from 10 borderline leprosy patients without reactions. Gene expression of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) type 2, which converts cortisol to cortisone, is down-regulated in skin from T1R lesions. However expression levels of 11beta-HSD type 1, which converts cortisone to cortisol, were similar in skin with and without reactions and did not change during anti-leprosy drug treatment. Prednisolone treatment of patients with reactions is associated with an upregulation of 11beta-HSD2 expression in skin. The down regulation of 11beta-HSD2 at the beginning of a reaction may be caused by pro-inflammatory cytokines in the leprosy reactional lesion and may be a local attempt to down-regulate inflammation. However in leprosy reactions this local response is insufficient and exogenous steroids are required to control inflammation.

Assessment of Free Drug Concentration in Cyclodextrin Formulations Is Essential to Determine Drug Potency in Functional In Vitro Assays.

Cyclodextrins (CD) have the ability to form inclusion complexes with drugs and can be used as excipients to enhance solubility of poorly soluble drugs. To make accurate estimations of the potency of the drug, knowledge of the free drug concentration is important. The aim of this study was to evaluate the applicability of calculated free drug concentrations toward response measurements in a transient receptor potential vanilloid receptor-1 cell-based in vitro assay. This included accounting for potential competitive CD binding of 2 transient receptor potential vanilloid receptor-1 active entities: 1 antagonist, and 1 agonist (capsaicin). Solubility of the CD-drug complexes was measured, and the ligand to substrate affinity in CD formulations was determined according to the phase-solubility technique. The total concentration of antagonist, agonist, CD, and the binding constants between ligands and CD were used to calculate the free concentration of CD ligands. For capsaicin and 2 of the 3 investigated model drugs, the calculated free drug concentration was consistent with the experimental in vitro data while it was overestimated for one of the compounds. In conclusion, the suggested approach can be used to calculate free drug concentration and competitive binding in CD formulations for the application of cell-based drug functionality assays.

Identification of ETV6-RUNX1-like and DUX4-rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia.

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

The genomic landscape of core-binding factor acute myeloid leukemias.

Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.

Capsaicin augments synaptic transmission in the rat medial preoptic nucleus.

The medial preoptic nucleus (MPN) is the major nucleus of the preoptic area (POA), a hypothalamic area involved in the regulation of body-temperature. Injection of capsaicin into this area causes hypothermia in vivo. Capsaicin also causes glutamate release from hypothalamic slices. However, no data are available on the effect of capsaicin on synaptic transmission within the MPN. Here, we have studied the effect of exogenously applied capsaicin on spontaneous synaptic activity in hypothalamic slices of the rat. Whole-cell patch-clamp recordings were made from visually identified neurons located in the MPN. In a subset of the studied neurons, capsaicin enhanced the frequency of spontaneous glutamatergic EPSCs. Remarkably, capsaicin also increased the frequency of GABAergic IPSCs, an effect that was sensitive to removal of extracellular calcium, but insensitive to tetrodotoxin. This suggests an action of capsaicin at presynaptic GABAergic terminals. In contrast to capsaicin, the TRPV4 agonist 4alpha-PDD did not affect GABAergic IPSCs. Our results show that capsaicin directly affects synaptic transmission in the MPN, likely through actions at presynaptic terminals as well as on projecting neurons. Our data add to the growing evidence that capsaicin receptors are not only expressed in primary afferent neurons, but also contribute to synaptic processing in some CNS regions.

The landscape of somatic mutations in infant MLL-rearranged acute lymphoblastic leukemias.

Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non-MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10(-5)) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Delineation of the proinflammatory cytokine cascade in fever induction.

Bacterial lipopolypolysaccharide (LPS)-induced fever involves induction of the proinflammatory cytokines interleukin (IL)-1 alpha, IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), and IL-6, both in the periphery and in the brain. These molecules can induce expression of each other and also regulate expression of their own receptors in a complex manner. The functional hierarchy of these highly inducible proteins is therefore difficult to determine. Using mice strains carrying the null mutations of IL-1 beta, IL-1RI, IL-1RAcP, or IL-6, respectively, we show that LPS-induced fever involves IL-1 beta, which acts at a complex consisting of the type I IL-1 receptor and the IL-1RAcP. This action occurs prior to central IL-6 release, which has been shown to be a necessary component of fever responses induced by LPS, IL-1 beta, and also TNF-alpha. In the absence of IL-1 beta, as in IL-1 beta-deficient mice, LPS, IL-1 alpha, and IL-1 beta cause hyperresponsive fevers when exogenously applied. Murine TNF-alpha is a poor pyrogen in mice even when mice are kept at thermoneutral temperature (30 degrees C). TNF-alpha-mediated fever depends on central IL-6 expression.

Integrated genetic and epigenetic analysis of childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

GSK-3 inhibition by an orally active small molecule increases bone mass in rats.

Glycogen synthase kinase 3ß (GSK-3ß) actions are central in the canonical Wnt pathway, important in many biological processes and a potential drug target for treating several diseases. It is appreciated that a balanced Wnt canonical signaling is crucial for the maintenance of normal bone mass. In this study we investigated the effects of a potent orally active GSK-3 inhibitor, AZD2858, on bone mass in rats. Treatment (1 µM) of human osteoblast cells with AZD2858 in vitro increased ß-catenin levels after a short period of time. In rats, oral AZD2858 treatment caused a dose-dependent increase in trabecular bone mass compared to control after a two-week treatment with a maximum effect at a dose of 20 mg/kg once daily (total BMC: 172% of control; p<0.001). A small but significant effect was also seen at cortical sites (total BMC: 111% of control; p<0.001). Biomechanical testing demonstrated an increase in both vertebral compression strength at a dose of 20 mg/kg once daily (Load at failure: 370% of control, p<0.001) and diaphyseal strength of femora subjected to a three point bending test (Load at failure: 115% of control; p<0.01). Furthermore, histomorphometry showed a dramatic increase in bone formation indices, and serum markers of both bone formation (Osteocalcin, 146% of control; p<0.001) and resorption (CTX, 189% of control; p<0.001) were elevated. Our conclusion is that a GSK-3 inhibitor drug may prove effective as an anabolic strategy in the treatment of diseases characterized by low bone mass, since AZD2858 has extensive bone building effects at predominantly trabecular sites.