Search

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. Significance: A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Supplementary Data from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Supplementary Figure from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.Significance:A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Povidone-iodide (Betadine) is an antiseptic that is applied topically and has many uses in the medical community, such as in wound care and pre- and post-operative surgical procedures. This study was done to measure the effectiveness of Betadine solutions in inhibiting the growth of Gram-negative and Gram-positive bacteria.The ability of 2.5 and 10% Betadine solutions to inhibit bacterial growth was measured againstAll the bacteria showed zones of inhibition. However, discs were also tested for live bacteria using the colony-forming unit assay. Complete killing was only seen forThis study showed several things. First, the zone of inhibition assay does not give an accurate assessment of antimicrobial properties when used alone and should be followed by a colony-forming unit assay. Second, 2.5% and 5% Betadine do not have effective antimicrobial properties against any of the bacteria tested, and 10% Betadine is only effective against

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-methylated GBM cell lines and patient-derived models was lethal and resulted in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, caused nuclear lobulation, blebbing and micronucleation, which subsequently resulted in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic VRK2 expression. Knockdown of VRK1 led to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Retinal hemorrhaging (RH) is indicative and prevalent in abusive head trauma (AHT)—yet the direct cause of the RH from AHT is unknown. Our hypothesis is that RH in AHT is the combination of shaking forces and hypertension. This combination of effects explains why RH is not normally observed in common childhood accidents but is nearly exclusively observed in AHT. An experimental model using porcine eyes was designed to ascertain the required pressure change for sudden RH and, via a computer model, the subsequent stress increase in blood vessels. The porcine eyes were cannulated via the maxillary artery and pressurized until perfusion and RH were observed. Fluid was injected into the head with a computer-controlled continuous flow syringe pump; video of the fundus was recorded during perfusion; and the pressure of the fluid entering the eye was recorded as well. A computer model was created in COMSOL to simulate loading from hypertension, shaking, and the combination of the forces. This model was validated via experimental data collected from the porcine model. It was found that hypertension or shaking alone did not cause an increase in stress required to cause RH. But when the loading of shaking and hypertension was combined, as would occur in AHT, the stress increases were greater than those extrapolated from the porcine model and would cause RH.

Introduction: Fibroblast growth factors (FGFs) are key mitogens in tissue homeostasis and cancer. FGF2 regulates self-renewal of multiple stem-cell types, is widely used in stem cell culture paradigms and has been adopted for cultivating the growth of cancer stem cells ex vivo. Research has shed light on the functions of FGF2 in brain tumors, particularly malignant glioma, and this has demonstrated that FGF2 increases self-renewal of glioblastoma stem cells.\udAreas covered: This review examines the potential targeting of FGF2 signaling as a possible treatment avenue for glioblastoma. The expression of FGF ligands and the FGFR family of receptor tyrosine kinases in the normal brain and in glioblastoma is described. Moreover, the paper sheds light on FGF/FGFR signaling, including the function of heparin/heparan sulfate proteoglycans in facilitating FGF signaling. We speculate on potential avenues for the therapeutic targeting of the FGF2-FGF receptor signaling axis in glioblastoma and the associated challenges envisioned with these approaches.\udExpert opinion: Precision targeting of FGF/FGFR signaling could improve prospective glioblastoma therapeutics and moderate adverse effects. Shrewd development of experimental models and FGF2 inhibitors could provide a ‘pharmacological toolbox’ for targeting diverse ligand/receptor combinations.

Aims Until recently, the pluripotency factor Octamer (ATGCAAAT)-binding transcriptional factor 4 (OCT4) was believed to be dispensable in adult somatic cells. However, our recent studies provided clear evidence that OCT4 has a critical atheroprotective role in smooth muscle cells. Here, we asked if OCT4 might play a functional role in regulating endothelial cell (EC) phenotypic modulations in atherosclerosis. Methods and results Specifically, we show that EC-specific Oct4 knockout resulted in increased lipid, LGALS3+ cell accumulation, and altered plaque characteristics consistent with decreased plaque stability. A combination of single-cell RNA sequencing and EC-lineage-tracing studies revealed increased EC activation, endothelial-to-mesenchymal transitions, plaque neovascularization, and mitochondrial dysfunction in the absence of OCT4. Furthermore, we show that the adenosine triphosphate (ATP) transporter, ATP-binding cassette (ABC) transporter G2 (ABCG2), is a direct target of OCT4 in EC and establish for the first time that the OCT4/ABCG2 axis maintains EC metabolic homeostasis by regulating intracellular heme accumulation and related reactive oxygen species production, which, in turn, contributes to atherogenesis. Conclusions These results provide the first direct evidence that OCT4 has a protective metabolic function in EC and identifies vascular OCT4 and its signalling axis as a potential target for novel therapeutics.

Synthetic lethality — a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone — can be co-opted for cancer therapeutics. A pair of paralog genes is among the most straightforward synthetic lethal interaction by virtue of their redundant functions. Here we demonstrate a paralog-based synthetic lethality by targeting Vaccinia-Related Kinase 1 (VRK1) in Vaccinia-Related Kinase 2 (VRK2)-methylated glioblastoma (GBM). VRK2 is silenced by promoter methylation in approximately two-thirds of GBM, an aggressive cancer with few available targeted therapies. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells results in decreased activity of the downstream substrate Barrier to Autointegration Factor (BAF), a regulator of post-mitotic nuclear envelope formation. VRK1 knockdown, and thus reduced BAF activity, causes nuclear lobulation, blebbing and micronucleation, which subsequently results in G2/M arrest and DNA damage. The VRK1-VRK2 synthetic lethal interaction is dependent on VRK1 kinase activity and is rescued by ectopic VRK2 expression. Knockdown of VRK1 leads to robust tumor growth inhibition in VRK2-methylated GBM xenografts. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC therapeutic resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. The knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. Mechanistically, SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown dramatically increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a novel GBM CSC-specific survival mechanism involving a previously uninvestigated cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of standard-of-care GBM therapies against therapeutically resistant CSCs.SummaryLauko et al. demonstrate a functional role for SerpinB3, which is elevated in glioblastoma cancer stem cells and protects against lysosomal-mediated cell death. SerpinB3 can be targeted to increase the efficacy of radiation in glioblastoma pre-clinical models.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSCin vitrogrowth and self-renewal as well asin vivotumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche.Graphical Abstract

The role of plasticity and epigenetics in shaping cancer evolution and response to therapy has taken center stage with recent technological advances including single cell sequencing. This roadmap article is focused on state-of-the-art mathematical and experimental approaches to interrogate plasticity in cancer, and addresses the following themes and questions: is there a formal overarching framework that encompasses both non-genetic plasticity and mutation-driven somatic evolution? How do we measure and model the role of the microenvironment in influencing/controlling non-genetic plasticity? How can we experimentally study non-genetic plasticity? Which mathematical techniques are required or best suited? What are the clinical and practical applications and implications of these concepts?

Glioblastoma (GBM) progression and resistance to conventional therapies is driven in part by cells within the tumor with stem cell properties including quiescence, self-renewal and drug efflux potential. It is thought that eliminating these cancer stem cells (CSCs) is a key component to successful clinical management of GBM. However, currently, few known molecular mechanisms driving CSCs can be exploited for therapeutic development. Core transcription factors such as SOX2, OLIG2, OCT4 and NANOG maintain the CSC state in GBM. Our laboratory recently uncovered a self-renewal signaling axis involving RBBP5 that is necessary and sufficient for CSC maintenance through driving expression of these core stem cell maintenance transcription factors. RBBP5 is a component of the WRAD complex, which promotes Lys4 methylation of histone H3 to positively regulate transcription. We hypothesized that targeting RBBP5 could be a means to disrupt epigenetic programs that maintain CSCs in stemness transcriptional states. We found that genetic and pharmacologic inhibition of the WRAD complex reduced CSC growth, self-renewal and tumor initiation potential. WRAD inhibitors partially dissembled the WRAD complex and reduced H3K4 trimethylation both globally and at the promoters of key stem cell maintenance transcription factors. Using a CSC reporter system, we demonstrated that WRAD complex inhibition decreased growth of SOX2/OCT4 expressing CSCs in a concentration-dependent manner as quantified by live imaging. Overall, our studies assess the function of the WRAD complex and the effect of WRAD complex inhibitors in preclinical models and specifically on the stem cell state for the first time in GBM. Studying the functions of the WRAD complex in CSCs may improve understanding of GBM pathogenesis and elucidate how CSCs survive despite aggressive chemotherapy and radiation. Our ongoing studies aim to develop brain penetrant inhibitors targeting the WRAD complex as an anti-CSC strategy that could potentially synergize with standard of care treatments.

Cellular heterogeneity is a hallmark of advanced cancers and has been ascribed in part to a population of self-renewing, therapeutically resistant cancer stem cells (CSCs). Glioblastoma (GBM), the most common primary malignant brain tumor, has served as a platform for the study of CSCs. In addition to illustrating the complexities of CSC biology, these investigations have led to a deeper understanding of GBM pathogenesis, revealed novel therapeutic targets, and driven innovation towards the development of next-generation therapies. While there continues to be an expansion in our knowledge of how CSCs contribute to GBM progression, opportunities have emerged to revisit this conceptual framework. In this review, we will summarize the current state of CSCs in GBM using key concepts of evolution as a paradigm (variation, inheritance, selection, and time) to describe how the CSC state is subject to alterations of cell intrinsic and extrinsic interactions that shape their evolutionarily trajectory. We identify emerging areas for future consideration, including appreciating CSCs as a cell state that is subject to plasticity, as opposed to a discrete population. These future considerations will not only have an impact on our understanding of this ever-expanding field but will also provide an opportunity to inform future therapies to effectively treat this complex and devastating disease.

The dressing material of a wound plays a key role since bacteria can live in the bandage and keep re-infecting the wound, thus a bandage is needed that blocks biofilm in the bandage. Using an in vivo wound biofilm model, we examined the effectiveness of an organo-selenium (OS)-coated polyester dressing to inhibit the growth of bacteria in a wound. Staphylococcus aureus (as well as MRSA, Methicillin resistant Staph aureus), Stenotrophomonas maltophilia, Enterococcus faecalis, Staphylococcus epidermidis, and Pseudomonas aeruginosa were chosen for the wound infection study. All the bacteria were enumerated in the wound dressing and in the wound tissue under the dressing. Using colony-forming unit (CFU) assays, over 7 logs of inhibition (100%) was found for all the bacterial strains on the material of the OS-coated wound dressing and in the tissue under that dressing. Confocal laser scanning microscopy along with IVIS spectrum in vivo imaging confirmed the CFU results. Thus, the dressing acts as a reservoir for a biofilm, which causes wound infection. The same results were obtained after soaking the dressing in PBS at 37 &deg, C for three months before use. These results suggest that an OS coating on polyester dressing is both effective and durable in blocking wound infection.

Betadine (Providone-Iodine) solution is a topically applied antiseptic, which has been used for wound care and surgery for decades for the prevention and treatment of skin and wound infections. However, several studies have documented the ineffectiveness of Betadine solution. Other topical antimicrobial dressings, including those that contain silver, have been used in the management of infected wounds. The present study was undertaken to determine if the combination of 5% Betadine solutions and silver colloidal gel (Ag-gel), is more effective than the individual materials in inhibiting the growth of both Gram negative and Gram positive bacteria. These determinations were carried out by both the colony forming unit (CFU) assay, and confocal laser scanning microscopy (CLSM). Ag-gel showed complete inhibition on all the bacteria, except Klebsiella pneumoniae CI strain while 5% Betadine concentrations did not completely kill any of the tested bacteria. However, K. pneumoniae was completely eliminated in the presence of the combination of 5% Betadine solution plus Ag-gel. Confocal laser microscopy confirmed the CFU results. Thus this study demonstrated that while the individual treatments are not effective in killing all the bacteria tested, the combination of 5% Betadine solution and Ag-gel completely kill all bacteria tested, including K. penumoniae CI.

Doxorubicin remains an essential component of many cancer regimens, but its use is limited by lethal cardiomyopathy, which has been difficult to target, owing to pleiotropic mechanisms leading to apoptotic and necrotic cardiac cell death. Here we show that BAX is rate-limiting in doxorubicin-induced cardiomyopathy and identify a small-molecule BAX inhibitor that blocks both apoptosis and necrosis to prevent this syndrome. By allosterically inhibiting BAX conformational activation, this compound blocks BAX translocation to mitochondria, thereby abrogating both forms of cell death. When co-administered with doxorubicin, this BAX inhibitor prevents cardiomyopathy in zebrafish and mice. Notably, cardioprotection does not compromise the efficacy of doxorubicin in reducing leukemia or breast cancer burden in vivo, primarily due to increased priming of mitochondrial death mechanisms and higher BAX levels in cancer cells. This study identifies BAX as an actionable target for doxorubicin-induced cardiomyopathy and provides a prototype small-molecule therapeutic.

IL1RAP is an emerging target for AML therapy. Studying its cell-intrinsic function revealed that IL1RAP interacts with and amplifies signaling through c-KIT and FLT3 in AML cells. This novel promiscuous role of IL1RAP in AML has implications for therapeutic targeting., The surface molecule interleukin-1 receptor accessory protein (IL1RAP) is consistently overexpressed across multiple genetic subtypes of acute myeloid leukemia (AML) and other myeloid malignancies, including at the stem cell level, and is emerging as a novel therapeutic target. However, the cell-intrinsic functions of IL1RAP in AML cells are largely unknown. Here, we show that targeting of IL1RAP via RNA interference, genetic deletion, or antibodies inhibits AML pathogenesis in vitro and in vivo, without perturbing healthy hematopoietic function or viability. Furthermore, we found that the role of IL1RAP is not restricted to the IL-1 receptor pathway, but that IL1RAP physically interacts with and mediates signaling and pro-proliferative effects through FLT3 and c-KIT, two receptor tyrosine kinases with known key roles in AML pathogenesis. Our study provides a new mechanistic basis for the efficacy of IL1RAP targeting in AML and reveals a novel role for this protein in the pathogenesis of the disease., Graphical Abstract

This work evaluates chronic safety in middle-aged ovariectomized rats supplemented with different dosages of green tea polyphenols (GTP) in drinking water. The experiment used 6-mo-old sham (n = 39) and ovariectomized (OVX, n = 143) female rats. All sham (n = 39) and 39 of the OVX animals received no GTP treatment and their samples were collected for outcome measures at baseline, 3 mo, and 6 mo (n = 13 per group for each). The remaining OVX animals were randomized into 4 groups receiving 0.15%, 0.5%, 1%, and 1.5% (n = 26 for each) of GTP (wt/vol), respectively, in drinking water for 3 and 6 mo. No mortality or abnormal treatment-related findings in clinical observations or ophthalmologic examinations were noted. No treatment-related macroscopic or microscopic findings were noted for animals administered 1.5% GTP supplementation. Throughout the study, there was no difference in the body weight among all OVX groups. In all OVX groups, feed intake and water consumption significantly decreased with GTP dose throughout the study period. At 6 mo, GTP intake did not affect hematology, clinical chemistry, and urinalysis, except for phosphorus and blood urea nitrogen (increased), total cholesterol, lactate dehydrogenase, and urine pH (decreased). This study reveals that the no-observed-adverse-effect level (NOAEL) of GTP is 1.5% (wt/vol) in drinking water, the highest dose used in this study.

Despite the identification of several oncogenic driver mutations leading to constitutive JAK–STAT activation, the cellular and molecular biology of myeloproliferative neoplasms (MPN) remains incompletely understood. Recent discoveries have identified underlying disease-modifying molecular aberrations contributing to disease initiation and progression. Here, we report that deletion of Nol3 (Nucleolar protein 3) in mice leads to an MPN resembling primary myelofibrosis (PMF). Nol3−/− MPN mice harbor an expanded Thy1+LSK stem cell population exhibiting increased cell cycling and a myelomonocytic differentiation bias. Molecularly, this phenotype is mediated by Nol3−/−-induced JAK–STAT activation and downstream activation of cyclin-dependent kinase 6 (Cdk6) and Myc. Nol3−/− MPN Thy1+LSK cells share significant molecular similarities with primary CD34+ cells from PMF patients. NOL3 levels are decreased in CD34+ cells from PMF patients, and the NOL3 locus is deleted in a subset of patients with myeloid malignancies. Our results reveal a novel genetic PMF-like mouse model and identify a tumor suppressor role for NOL3 in the pathogenesis of myeloid malignancies.

Therapies targeting cell-surface antigens in acute myeloid leukemia (AML) have been tested over the past 20 years with limited improvement in overall survival. Recent advances in the understanding of AML pathogenesis support therapeutic targeting of leukemia stem cells as the most promising avenue toward a cure. In this review, we provide an overview of the evolving leukemia stem cell (LSC) model, including evidence of the cell of origin, cellular and molecular disease architecture, and source of relapse in AML. In addition, we explore limitations of current targeted strategies utilized in AML and describe the various immunophenotypic antigens that have been proposed as LSC-directed therapeutic targets. We draw lessons from current approaches as well as from the (pre)-LSC model to suggest criteria that immunophenotypic targets should meet for more specific and effective elimination of disease-initiating clones, highlighting in detail a few targets that we suggest fit these criteria most completely.

In this review article, we aimed to analyze the available data on the ocular manifestations of von Hippel-Lindau (VHL) disease. In this disease, the VHL protein becomes inactivated by germline mutations of the VHL tumor suppressor gene on chromosome 3p25-26, resulting in an overproduction of VEGF in non-hypoxic conditions. Ocular manifestations are expected in roughly half of VHL patients. Retinal capillary hemangioblastomas (RCHs) are the most commonly observed tumors in VHL and are often the initial manifestation of the disease. Ablative therapy, surgical resection, and pharmacotherapy have been implemented to control tumors. Left untreated, RCHs will often enlarge, emphasizing the importance of early diagnosis and treatment to preserve vision. Complications of enlarging peripheral or optic nerve tumors may be severe. Large RCHs may disrupt normal retinal architecture, eventually leading to exudative retinal detachment. Rarely, non-retinal manifestations, such as neovascularization of the iris or cornea, may progress to neovascular glaucoma and vision loss. Ablative therapy of larger tumors carries increasing risks and offers limited success, often necessitating surgical resection. Because this life-threatening disease is not routinely encountered in clinical practice, clinicians will benefit from our review which brings awareness to the ocular presentation of VHL and lifelong screening recommendations for diagnosed patients.

IMPORTANCE: Although almost equal numbers of male and female medical students enter into ophthalmology residency programs, whether they have similar surgical experiences during training is unclear. OBJECTIVE: To determine differences for cataract surgery and total procedural volume between male and female residents during ophthalmology residency. DESIGN, SETTING, PARTICIPANTS: This retrospective, longitudinal analysis of resident case logs from 24 US ophthalmology residency programs spanned July 2005 to June 2017. A total of 1271 residents were included. Data were analyzed from August 12, 2017, through April 4, 2018. MAIN OUTCOMES AND MEASURES: Variables analyzed included mean volumes of cataract surgery and total procedures, resident gender, and maternity or paternity leave status. RESULTS: Among the 1271 residents included in the analysis (815 men [64.1%]), being female was associated with performing fewer cataract operations and total procedures. Male residents performed a mean (SD) of 176.7 (66.2) cataract operations, and female residents performed a mean (SD) of 161.7 (56.2) (mean difference, −15.0 [95% CI, −22.2 to −7.8]; P

The tumor suppressor p53 is often inactivated via its interaction with endogenous inhibitors mouse double minute 4 homolog (MDM4 or MDMX) or mouse double minute 2 homolog (MDM2), which are frequently overexpressed in patients with acute myeloid leukemia (AML) and other cancers. Pharmacological disruption of both of these inter-actions has long been sought after as an attractive strategy to fully restore p53-dependent tumor suppressor activity in cancers with wild-type p53. Selective targeting of this pathway has thus far been limited to MDM2-only small-molecule inhibitors, which lack affinity for MDMX. We demonstrate that dual MDMX/MDM2 inhibition with a stapled a-helical peptide (ALRN-6924), which has recently entered phase I clinical testing, produces marked antileukemic effects. ALRN-6924 robustly activates p53-dependent transcription at the single-cell and single-molecule levels and exhibits biochemical and molecular biological on-target activity in leukemia cells in vitro and in vivo. Dual MDMX/MDM2 inhibition by ALRN-6924 inhibits cellular proliferation by inducing cell cycle arrest and apoptosis in cell lines and primary AML patient cells, including leukemic stem cell-enriched populations, and disrupts functional clonogenic and serial replating capacity. Furthermore, ALRN-6924 markedly improves survival in AML xenograft models. Our study provides mechanistic insight to support further testing of ALRN-6924 as a therapeutic approach in AML and other cancers with wild-type p53.

Stanley et al. show that loss of Nol3 in mice leads to a myeloproliferative phenotype resembling primary myelofibrosis, with activation of JAK–STAT signaling and significant cellular and molecular resemblance to human disease. These findings provide a novel role for Nol3 in hematopoiesis and myeloid malignancies., Despite the identification of several oncogenic driver mutations leading to constitutive JAK–STAT activation, the cellular and molecular biology of myeloproliferative neoplasms (MPN) remains incompletely understood. Recent discoveries have identified underlying disease-modifying molecular aberrations contributing to disease initiation and progression. Here, we report that deletion of Nol3 (Nucleolar protein 3) in mice leads to an MPN resembling primary myelofibrosis (PMF). Nol3−/− MPN mice harbor an expanded Thy1+LSK stem cell population exhibiting increased cell cycling and a myelomonocytic differentiation bias. Molecularly, this phenotype is mediated by Nol3−/−-induced JAK–STAT activation and downstream activation of cyclin-dependent kinase 6 (Cdk6) and Myc. Nol3−/− MPN Thy1+LSK cells share significant molecular similarities with primary CD34+ cells from PMF patients. NOL3 levels are decreased in CD34+ cells from PMF patients, and the NOL3 locus is deleted in a subset of patients with myeloid malignancies. Our results reveal a novel genetic PMF-like mouse model and identify a tumor suppressor role for NOL3 in the pathogenesis of myeloid malignancies.

Resistance to apoptosis is a hallmark of human cancer. The BCL-2 protein family includes both pro- and anti-apoptotic proteins and has a central role in regulating mitochondrial apoptosis. Cancer cells most frequently ensure their survival and resistance to treatments by overexpression of anti-apoptotic BCL-2 proteins to maintain BAX and other pro-apoptotic members suppressed. Pro-apoptotic BAX is the cardinal executioner BCL-2-family member that, upon conformational activation and oligomerization at the mitochondrial outer membrane (MOM), causes permeabilization of the MOM and release of mitochondrial factors, e.g., cytochrome c, that activate the caspase cascade of apoptosis. Interestingly, the vast majority of cancer cells contain functional BAX in an inactive conformation or suppressed by anti-apoptotic proteins; mutations or alterations in BAX that may cause its inactivation to occur at a low frequency. Therefore, we hypothesized that induction of apoptosis by direct activation of BAX offers the possibility of a new anticancer strategy. Using a structure-based drug design approach we developed BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site (trigger site) and induces conformational changes to BAX, resulting in BAX mitochondrial translocation, oligomerization and mitochondrial dysfunction (Reyna et al., Cancer Cell 2017). BTSA1 induces activation of cytosolic BAX, leading to prompt and robust mitochondrial apoptosis in acute myeloid leukemia (AML) cell lines. The efficacy of BTSA1 is regulated by the availability of anti-apoptotic BCL-2 proteins to inhibit activated BAX and therefore, higher levels of cytosolic BAX monomer correlated with higher efficacy of BAX-mediated mitochondrial dysfunction. BTSA1-induced BAX activation promotes effectively apoptosis in primary AML samples and has significant antitumor activity in human AML xenograft models, increasing host survival. BTSA1 is orally bioavailable with excellent pharmacokinetics at therapeutically effective doses; it does not show any detectable toxicity in the hematopoietic system or other tissues. Importantly, BTSA1 is also effective in inducing apoptosis in leukemic stem cell-enriched fractions (CD34+CD38−) while sparing healthy counterparts. Consistent with their sensitivity to direct BAX activation, AML blasts and highly purified stem and progenitor cell populations from AML patients when compared to healthy counterparts displayed higher expression of BAX. Overall, our data provide proof of concept for BAX as a druggable target and demonstrate the therapeutic potential of direct BAX activation by BTSA1 as an effective treatment strategy in AML. Citation Format: Denis Reyna, Thomas Garner, Andrea Lopez, Felix Kopp, Gaurav Choudhary, Ashwin Sridharan, Swathi-Rao Narayanagari, Kelly Mitchell, Baoxia Dong, Boris Bartholdy, Loren Walensky, Amit Verma, Ulrich Steidl, Evripidis Gavathiotis. Direct small-molecule BAX activation in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 951.

The project is to evaluate chronic toxicity and safety in middle-aged ovariectomized rats supplemented with different dosages of green tea polyphenols (GTP) in drinking water. 182 SD were sham (n=3...

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Functionally critical decreases in levels or activity of the ETS family transcription factor PU.1 are present in approximately 2/3 of patients with acute myeloid leukemia (AML), across different AML subtypes (Sive, Leukemia 2016) including at the stem cell level (Steidl, Nat Genet 2006; Will, Nat Med 2015). Thus, targeting PU.1 could be an appealing option for treatment. As complete loss of PU.1 leads to stem cell failure (Iwasaki, Blood 2005), we hypothesized that PU.1 inhibition could eradicate leukemic cells harboring already low levels of PU.1, with modest effects on normal cells. We initially tested this hypothesis using 3 different shRNAs, and found that PU.1 inhibition led to a significant decrease in proliferation and clonogenicity, and increased apoptosis of mouse and human leukemic cell lines with low PU.1 levels, as well as the majority of primary human AML cells tested. We demonstrated that these effects were indeed due to decreased PU.1 levels by retroviral add-back experiments. The direct pharmacologic targeting of transcription factors has proven challenging in the past. Besides the core ETS binding motif (GGAA) in the DNA major groove, PU.1 binding to chromatin depends on additional minor groove contacts enriched for AT nucleotides upstream of the ETS motif, which determine selectivity for PU.1. Using an integrated screening strategy utilizing biosensor surface plasmon resonance, DNA footprinting, and cell-based dual-color PU.1 reporter assays, we developed novel small molecules of the heterocyclic diamidine family acting as first-in-class PU.1 inhibitors. Targeted occupancy by our compounds in the minor groove induces perturbations in DNA conformation that are transmitted to the PU.1 site in the major groove and thus inhibits PU.1 binding via an allosteric mechanism. Consistent with this, the inhibitory effects were selective for PU.1 versus other ETS transcription factors. Treatment with 3 different compounds led to cell growth inhibitory effect with respect to PU.1 level and preferentially affects PU.1low AML cells. Similarly to what we observed with shRNAs, treatment with our novel inhibitors led to decreased proliferation and colony forming capacity, increased apoptosis, and disrupted serial replating capacity of PU.1low AML cells and a majority of primary AML cell samples. Targeted ChIP and expression analysis showed that the compounds disrupt PU.1-promoter interaction and lead to downregulation of canonical PU.1 transcriptional targets in AML cells, confirming on-target activity in AML cells. Genome-wide analysis showed highly significant enrichment of known transcriptional targets of PU.1, and selectivity over genes regulated by other ETS family members. Comparison with published transcriptomic and PU.1 ChIP-seq data sets, as well as ARACNe analysis of the PU.1 regulon in primary AML cells, demonstrated that the inhibitors antagonize PU.1-regulated pathways at a genome-wide level. ChIP-seq performed in PU.1low AML cells confirmed a genome-wide decrease of PU.1 peaks after treatment and provides novel insight into the molecular mechanisms mediating the anti-leukemic effects of pharmacological PU.1 inhibition. To test the effects of PU.1 inhibition on normal hematopoiesis, we treated normal hematopoietic stem/progenitors cells (HSPC) in colony forming assays and saw decreased production of mature granulo-monocytic cells, consistent with PU.1's known role in this lineage. However, this effect was reversible upon drug removal, and serial replating capacity was not affected suggesting no significant effects on more immature HSPC. Congenic transplantation assays of treated normal bone marrow cells led to no change in myeloid and T-cells and only a modest decrease in B-cell numbers. Lastly, in vivo treatment with PU.1 inhibitors in mouse and human AML (xeno)transplantation models significantly decreased tumor burden and increased survival. To conclude, our study provides proof-of-principle for PU.1 inhibition as a novel therapeutic strategy in AML. Furthermore, we present the development of first-in-class PU.1 inhibitors acting via an allosteric minor groove-mediated mechanism. Our work shows that the specific pharmacological targeting of the DNA interaction of transcription factors such as PU.1 is feasible in principle, and may open the way for targeting of other transcription factors through minor groove-directed approaches. Disclosures Will: Novartis Pharmaceuticals: Consultancy, Research Funding. Steidl: Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Bayer Healthcare: Consultancy.

The BCL-2 family protein BAX is a central mediator of apoptosis. Overexpression of anti-apoptotic BCL-2 proteins contributes to tumor development and resistance to therapy by suppressing BAX and its activators. We report the discovery of BTSA1, a pharmacologically optimized BAX activator that binds with high affinity and specificity to the N-terminal activation site and induces conformational changes to BAX leading to BAX-mediated apoptosis. BTSA1-induced BAX activation effectively promotes apoptosis in leukemia cell lines and patient samples while sparing healthy cells. BAX expression levels and cytosolic conformation regulate sensitivity to BTSA1. BTSA1 potently suppressed human acute myeloid leukemia (AML) xenografts and increased host survival without toxicity. This study provides proof-of-concept for direct BAX activation as a treatment strategy in AML.

Purpose Biofilm formation is a problem for solid and sponge-type scleral buckles. This can lead to complications that require removal of the buckle, and result in vision loss due to related ocular morbidity, primarily infection, or recurrent retinal detachment. We investigate the ability of a covalent organo-selenium coating to inhibit biofilm formation on a scleral buckle. Methods Sponge and solid Labtican brand scleral buckles were coated with organo-selenium coupled to a silyation reagent. Staphylococcus aureus biofilm formation was monitored by a standard colony-forming unit assay and the confocal laser scanning microscopy, while Pseudomonas aeruginosa biofilm formation was examined by scanning electron microscopy. Stability studies were done, by soaking in phosphate buffer saline (PBS) at room temperature for 2 months. Toxicity against human corneal epithelial cell was examined by growing the cells in the presence of organo-selenium–coated scleral buckles. Results The organo-selenium coating inhibited biofilm formation by gram-negative and gram-positive bacteria. The buckle coatings also were shown to be fully active after soaking in PBS for 2 months. The organo-selenium coatings had no effect on the viability of human corneal epithelial cells. Conclusions Organo-selenium can be used to covalently coat a scleral buckle, which is stable and inhibits biofilm formation for gram-negative and gram-positive bacteria. The organo-selenium buckle coating was stable and nontoxic to cell culture. Translational Relevance This technology provides a means to inhibit bacterial attachment to devices attached to the eye, without damage to ocular cells.

This Final EIS has been prepared in accordance with the National Environmental Policy Act (NEPA). The public and agency scoping process resulted in the analysis of the following environmental resources: noise, air quality, safety, soils and water, biological resources, cultural resources, land use, infrastructure and transportation, hazardous materials and wastes, socioeconomics, and environmental justice and the protection of children. The Secretary of the Air Force proposes to beddown KC-46A aircraft for MOB 2 at one of five alternative locations. The goal of KC-46A basing and fielding is to continue to provide optimum Combatant Commander support and to efficiently meet regional and global receiver demands while replacing the KC-135 fleet. The ANG host unit would be assigned principal responsibility of the physical resources for mission accomplishment (aircraft, equipment, facilities) and the active duty associate unit would share those resources. Five alternative ANG locations were selected for this beddown: * Forbes Air National Guard Station (ANGS), Kansas; * Joint Base McGuire-Dix-Lakehurst (JB MDL), New Jersey; * Pease ANGS, New Hampshire; * Pittsburgh ANGS, Pennsylvania; and, * Rickenbacker ANGS, Ohio. The USAF has identified Pease ANGS as the preferred alternative.

Objective To ascertain the sources of community pharmacists' knowledge base for counter-prescribing in pregnancy, whether sufficient additional information was readily accessible and, if not, to discover pharmacists' areas of concern. Method A structured telephone interview of community pharmacists by a single researcher using a pre-piloted questionnaire to obtain both quantitative and qualitative data. Questions covered previous education about counter-prescribing in pregnancy, reference sources used, and satisfaction with available reference sources. Respondents were also asked to cite examples where they would or would not recommend a medicine in pregnancy. Setting A random sample of 50 community pharmacies in Scotland. Key findings From the high response rate (43 pharmacists, 86 per cent) the topic was demonstrably important to community pharmacists, most of whom perceived their knowledge base for counter-prescribing in pregnancy to be experience-gained. Thirty-four pharmacists (79 per cent) agreed there was a need for more information to be available. Ten pharmacists (23 per cent) highlighted complementary therapies as an area where they found available information inadequate. Examples of requests from pregnant women and the action taken by the pharmacist are reported. Conclusion There is a need for an accurate, current and comprehensive data source for counter-prescribing in pregnancy. The field of complementary medicines and therapies is of particular concern to many community pharmacists. More research is required into GP referrals.

Human umbilical cord blood (CB)-derived unrestricted somatic stem cells (USSCs) have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. Here we demonstrated that USSCs could be induced to express genes that hallmark keratinocyte differentiation. We also demonstrated that USSCs express type VII collagen (C7), a protein that is absent or defective in patients with an inherited skin disease, recessive dystrophic epidermolysis bullosa (RDEB). In mice with full-thickness excisional wounds, a single intradermal injection of USSCs at a 1-cm distance to the wound edge resulted in significantly accelerated wound healing. USSC-treated wounds displayed a higher density of CD31+ cells, and the wounds healed with a significant increase in skin appendages. These beneficial effects were demonstrated without apparent differentiation of the injected USSCs into keratinocytes or endothelial cells. In vivo bioluminescent imaging (BLI) revealed specific migration of USSCs modified with a luciferase reporter gene, from a distant intradermal injection site to the wound, as well as following systemic injection of USSCs. These data suggest that CB-derived USSCs could significantly contribute to wound repair and be potentially used in cell therapy for patients with RDEB.

Introduction: The omentum is the primary site of metastasis in both ovarian cancer models and clinical disease. It is composed predominantly of adipose studded with lymphoreticular organs (milky spots), distinguishing it from other peritoneal adipose. Milky spots are specialized for immune cell trafficking and peritoneal surveillance. We and others have shown that ovarian cancer cells exploit the physiologic function(s) of these structures for omental metastatic colonization. The purpose of this study was to identify cellular and molecular mechanisms responsible for ovarian cancer homing to and growth within milky spot structures. Experimental Procedures: Quantitative in vivo and ex vivo assays were used to evaluate human (SKOV3ip.1, HEYA8, and CaOV3) and murine (ID8) ovarian cancer cell localization to milky spots on the murine omental fat band. Assays were conducted using C57/Bl6 mice or those lacking B cells (Igh6-/-); T cells (Nude); B and T cells (Rag1-/-); or B, T, and NK cells (BN XID). In vitro assays were used to assess the migration-promoting activity of omentum- and macrophage-conditioned media. Standard approaches were used to assess protein expression, cell growth and viability, etc. Rationale: Milky spots provide resident tissue macrophages and lymphocytes needed for peritoneal homeostasis. Macrophages and stromal cells secrete chemokines promoting peritoneal lymphocyte homing to the omentum. In response to peritoneal irritants, activated CD11b+ milky spot macrophages organize coordinated expansion of vascular and stromal compartments. The increase in both the number and size of milky spots is needed to process particulates, resolve infections, and encapsulate foreign bodies. Hypothesis: CD11b+ cells secrete homeostatic chemokines promoting G-protein-dependent migration of ovarian cancer cells to milky spots. Ovarian cancer cell binding to adhesion molecules on the milky spot surface activates CD11b+ cell-dependent tissue remodeling, creating a microenvironment promoting ovarian cancer growth. New Findings: Consistent with our hypothesis, in vivo assays showed that macrophage depletion prior to injection of ID8 and SKOV3ip.1 cells prevented microscopic metastasis formation. In vitro assays found that macrophages are required for ovarian cancer cell localization to milky spots. These data prompt the hypothesis that CD11b+ cells produce a factor(s) responsible for the migration-promoting ability of omentum-conditioned media. To test this, media was conditioned by omental adipose isolated from mice after macrophage depletion. In support of our hypothesis, the migration-promoting activity of macrophage-depleted omentum-conditioned media was on a par with that of media conditioned by milky spot-deficient adipose. Further, media conditioned by CD11b+ cells recapitulates the migration-promoting activity exhibited by omentum-conditioned media. In vitro and ex vivo assays were used to test whether ovarian cancer cells utilize mechanisms analogous to lymphocyte trafficking for milky spot homing. Specifically, cells were pretreated with pertussis toxin or vehicle alone, and then evaluated for migration in response to omentum-conditioned media and milky spot localization ex vivo. Consistent with data reported for lymphocyte homing, pertussis toxin pre-treatment caused a 40% to 50% reduction in ovarian cancer cell homing. Conclusions and Current Efforts: Our data support a model in which CD11b+ macrophages secrete one or more chemokines promoting G-protein receptor-dependent ovarian cancer cell migration, and potentially integrin activation, which mediate milky spot homing. Current experiments focus on identification of integrin-ligand interactions, CD11b+ cell activation, and defining the link between ovarian cancer cell growth and increase in milky spot size and number. Citation Format: Venkatesh Krishnan, Kelly Mitchell, Jason Miska, Sophia George, Patricia Shaw, Victoria Seewaldt, Maciej Lesniak, Marina Chekmareva, Lev Becker, Vinita Parkash, Cindy Miranti, Carrie Rinker-Schaeffer. Ovarian cancer cells hijack immune functions of omental milky spots for metastatic colonization. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A82.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival". Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers. Disclosures Gao: GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. AML is hallmarked by a differentiation block and patient outcomes remain poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. Hence the development of novel targeted therapies for treatment of AML subtypes are required. Of note, inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Treatment of primary IDH1 mutant AML patients' cells ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in leukemic blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including “Cell Cycle”, “G1/S transition”, “Cellular growth and proliferation”, and “Cell death and survival”. Taken together, our findings provide novel insight into the effects of inhibition of mutant IDH1 in primary AML patients' cells and open avenues for future investigations with these and other novel allosteric inhibitors for targeting IDH1 mutants in leukemia and possibly in other cancers. Citation Format: Ujunwa C. Okoye-Okafor, Boris Bartholdy, Jessy Cartier, Enoch Gao, Beth Pietrak, Alan R. Rendina, Cynthia Rominger, Chad Quinn, Angela Smallwood, Ken Wiggall, Alexander Reif, Stan Schmidt, Hongwei Qi, Huizhen Zhao, Gerard Joberty, Maria Faelth-Savitski, Marcus Bantscheff, Gerard Drewes, Chaya Duraiswami, Pat Brady, Swathi-Rao Narayanagari, Ileana Antony-Debre, Kelly Mitchell, Heng Rui Wang, Yun-Ruei Kao, Maximilian Christopeit, Luis Carvajal, Laura Barreyro, Elisabeth Paietta, Britta Will, Nestor Concha, Nicholas D. Adams, Benjamin Schwartz, Michael T. McCabe, Jaroslav Maciejewski, Amit Verma, Ulrich Steidl. Novel allosteric IDH1 mutant Inhibitors for differentiation therapy of acute myeloid leukemia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C38.

Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS and cure rates remain below 20%. AML/MDS arise following the accumulation of stepwise genetic and epigenetic changes in hematopoietic stem and progenitor cells (HSPC). Utilizing a novel strategy of parallel transcriptional analysis of sorted HSPC populations in distinct subtypes of AML, we compared the gene expression in AML HSPC with identical compartments from age-matched healthy controls and identified Interleukin 1 receptor accessory protein (IL1RAP) as one of the most significantly upregulated genes in HSPC in all examined subtypes of AML. Fluorescence in situ hybridization of sorted IL1RAP+ and IL1RAP- cells from patients with monosomy 7 AML (-7) indicated that the aberrant clone was restricted to IL1RAP+ cells, demonstrating that IL1RAP overexpression is a distinguishing feature of the -7 clone. Multivariate analysis of a large cohort of patients with normal karyotype AML showed that patients with high IL1RAP levels had inferior overall survival than patients with lower IL1RAP levels, suggesting an independent prognostic value for this molecule in AML. IL1RAP expression levels in MDS were found elevated on stem cells of patients with high risk disease, proposing a role of IL1RAP in higher risk MDS and progression to AML. Downregulation of IL1RAP expression by lentivirally expressed shRNAs decreased clonogenicity in cell lines and AML/MDS primary patient samples, induced apoptosis of AML cells, and reduced proliferation of AML cells and infiltration of hematopoietic organs in vivo. IL1RAP is a transmembrane protein required for signaling through several receptors of the IL1 family. Downregulation of IL1RAP expression in AML cells led to phosphorylation changes in several kinases and their substrates suggesting participation of IL1RAP in multiple signaling pathways and highlighting its potential as therapeutic target. We investigated whether inhibition of IL1RAP with pharmacological compounds is feasible and effective. Antibody-mediated inhibition of IL1RAP led to inhibition of AML cell growth in vitro. In addition, we designed peptides to interfere with IL1RAP-receptor interactions which lead to inhibition of AML cell growth. Both types of agents are being further tested and optimized. In summary, our study reveals IL1RAP as aberrantly expressed on HSPC of AML and high-risk MDS patients. Inhibition of IL1RAP is feasible and functionally effective, and thus has the potential to lead to novel therapies specifically directed at such stem cells. Beyond IL1RAP, our study provides a map of dysregulated transcripts in HSPC from patients with AML, which may offer further opportunities for therapeutic intervention. The strategy of comparative analysis of sorted stem and progenitor cells in cancer versus healthy controls may be applicable to other type of cancers with a suspected stem cell origin, and instrumental for the identification of targets for stem cell-directed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C225. Citation Format: Laura Barreyro, Kelly Mitchell, Britta Will, Boris Bartholdy, Li Zhou, Tihomira Todorova, Robert Stanley, Susana Ben-Neriah, Cristina Montagna, Samir Parekh, Andrea Pellagatti, Jacqueline Boultwood, Elisabeth Paietta, Rhett Ketterling, Larry Cripe, Hugo Fernandez, Peter Greenberg, Jacob Rowe, Martin Tallman, Christian Steidl, Constantine Mitsiades, Amit Verma, Ulrich Steidl. IL1RAP as functionally relevant target for stem-cell directed therapy in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C225.