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Autologous immune cell therapies are potentially curative, but cost and manufacturing complexity limit access. Off-the-shelf therapies may address these gaps, but further development is needed. The NK92 cell line has a natural killer-like phenotype, has efficacy in cancer clinical trials, and is safe after irradiation. However, NK92 lose activity post-injection, limiting efficacy. This may be addressed by engineering NK92 to express stimulatory factors, for which comparative analysis is needed. Thus, we systematically explored expression of synthetic cytokines for enhancing NK92 production and performance. All synthetic cytokines evaluated (membrane-bound IL2 and IL15, and engineered versions of Neoleukin-2/15, IL15, IL12, and decoy resistant IL18) enhanced NK92 cytotoxicity. Genetically modified cells preferentially expanded by expressing membrane-bound but not soluble synthetic cytokines, and all engineered cells remained sensitive to irradiation. Interestingly, some membrane-bound cytokines conferred cell-contact independent paracrine activity partly attributable to extracellular vesicles. Finally, we characterized interactions within consortia of differently engineered NK92.

Solution-processable [1]benzothieno[3,2-b]benzothiophene (BTBT) derivatives with various end-capping groups, 2-(phenylethynyl)benzo[b]benzo[4,5]thieno[2,3-d]thiophene (Compound 1), 2-octyl-7-(5-(phenylethynyl)thiophen-2-yl)benzo[b]benzo[4,5]thieno[2,3-d]thiophene (Compound 2), and triisopropyl((5-(7-octylbenzo[b]benzo[4,5]thieno[2,3-d]thiophen-2-yl)thiophen-2-yl)ethynyl)silane (Compound 3), have been synthesized and characterized as active layers for organic field-effect transistors (OFETs). Thermal, optical, and electrochemical properties of the newly synthesized compounds were characterized using thermogravimetric analysis (TGA), a differential scanning calorimeter (DSC), UV–vis spectroscopy, and cyclic voltammetry (CV). Thin films of each compound were formed using the solution-shearing method and the thin film surface morphology and texture of the corresponding films were characterized using atomic force microscopy (AFM) and θ–2θ X-ray diffraction (XRD). All semiconductors exhibited p-channel characteristics in ambient and Compound 1 showed the highest electrical performance with a carrier mobility of ~0.03 cm2/Vs and current on/off ratio of ~106.

The CD8+ T cells within tumor microenvironment are the most essential component in tumor immunity. Activation of CD8+ T cells is coupled to various metabolic reprogramming. Glutathione (GSH) as the key role in oxidative metabolismto buffer increased reactive oxygen species (ROS) in activated T cells, is important for T cells effector functions in the context of inflammation. The function and mechanism by GSH metabolism in regulating anti-tumor immunity of CD8+ T cells remain unknown. Here, we show that GSH is dependent on glutathione peroxidase 4 (Gpx4) to maintain CD8+ T cell activity, and A2AR signaling pathway interacts with the GSH-Gpx4 axis to reprogram the metabolism and survival of functional CD8+ T cells. Interestingly, A2AR signaling blockade increases effector function of antitumor CD8+ T cells, but facilitates the assumption of intracellular GSH, leading to rapid ferroptosis. Notably, combination treatment with a potent ferroptosis inhibitor liproxstatin-1 (Lip-1) and A2AR antagonists elicits a synergistic antitumor efficacy and enhanced mitochondrial functionality of antitumor CD8+ T cells in multiple mouse tumor models. Finally, we generate a gene expression signature for GSH metabolism in tumor-infiltrating CD8+ T cells positively correlating with favorable clinical outcomes. Our work demonstrate a critical role of GSH metabolism in modulating antitumor CD8+ T cell survival and functionality, pointing to new strategies of targeting these cells for cancer immunotherapy.

Activating the immune co-stimulatory receptor 4-1BB (CD137) with agonist antibody binding and crosslinking-inducing agents that elicit 4-1BB intracellular signaling potentiates the anti-tumor responses of CD8+ T cells. However, the underlying in-depth mechanisms remain to be defined. The type 2 adenosine receptors (primarily the high affinity receptor, A2AR) predominantly expressed on CD8+ T cells inhibit T cell activation and expansion by blocking TCR signaling in a cAMP-dependent manner. Here, we show that inactivation of the low affinity receptor A2BR rather than A2AR by continuous treatment of antagonists and/or genetic deletion induces superior survival advantage of effector CD8+ T cells with agonistic 4-1BB co-stimulation especially upon chronic TCR stimulation and/or long-term antigen exposure. Mechanistically, A2BR inactivation helps sustain the increased energy and biosynthetic requirements through the accumulation of intracellular glutathione (GSH) in response to agonistic 4-1BB co-stimulation. Importantly, A2BR inactivation in combination with agonistic 4-1BB co-stimulation displays a greater ability to modulate mitochondrial fitness for anti-tumor CD8+ T cell expansion while minimize T cell exhaustion. Thus, the A2BR pathway plays an unexpected role in metabolic reprogramming of GSH upon agonistic 4-1BB co-stimulation that allows the fine-tuning of the anti-tumor responses of CD8+ T cells. Supported by NCI CCSG P30 CA060553 R01CA258857

Genetic studies have identified recurrent somatic mutations in acute myeloid leukemia (AML) patients, including in the Wilms’ tumor 1 (WT1) gene. The molecular mechanisms by which WT1 mutations contribute to leukemogenesis have not yet been fully elucidated. We investigated the role of Wt1 gene dosage in steady-state and pathologic hematopoiesis. Wt1 heterozygous loss enhanced stem cell self-renewal in an age-dependent manner, which increased stem cell function over time and resulted in age-dependent leukemic transformation. Wt1-haploinsufficient leukemias were characterized by progressive genetic and epigenetic alterations, including those in known leukemia-associated alleles, demonstrating a requirement for additional events to promote hematopoietic transformation. Consistent with this observation, we found that Wt1 depletion cooperates with Flt3-ITD mutation to induce fully penetrant AML. Our studies provide insight into mechanisms of Wt1-loss leukemogenesis and into the evolutionary events required to induce transformation of Wt1-haploinsufficient stem/progenitor cells.

Patients with myeloproliferative neoplasms (MPNs) frequently progress to bone marrow failure or acute myeloid leukemia (AML), and mutations in epigenetic regulators such as the metabolic enzyme isocitrate dehydrogenase (IDH) are associated with poor outcomes. Here, we showed that combined expression of Jak2V617Fand mutant IDH1R132Hor Idh2R140Q induces MPN progression, alters stem/progenitor cell function, and impairs differentiation in mice. Jak2V617FIdh2R140Q–mutant MPNs were sensitive to small-molecule inhibition of IDH. Combined inhibition of JAK2 and IDH2 normalized the stem and progenitor cell compartments in the murine model and reduced disease burden to a greater extent than was seen with JAK inhibition alone. In addition, combined JAK2 and IDH2 inhibitor treatment also reversed aberrant gene expression in MPN stem cells and reversed the metabolite perturbations induced by concurrent JAK2 and IDH2 mutations. Combined JAK2 and IDH2 inhibitor therapy also showed cooperative efficacy in cells from MPN patients with both JAK2mutand IDH2mutmutations. Taken together, these data suggest that combined JAK and IDH inhibition May offer a therapeutic advantage in this high-risk MPN subtype., Damon Runyon Cancer Research Foundation (DRG-2241-15), Howard Hughes Medical Institute (Faculty Scholars Award), Stand Up To Cancer, National Cancer Institute (U.S.) (P50CA165962), National Cancer Institute (U.S.) (P30CA14051), Koch Institute for Integrative Cancer Research ( Dana-Farber Harvard Cancer Center Bridge Project), Leukemia & Lymphoma Society of America. Specialized Center of Research (SCOR) Program, National Institutes of Health (U.S.) (grant U54OD020355-01), National Institutes of Health (U.S.) (grant NCI R01CA172636), National Institutes of Health (U.S.) (grant R35CA197594), National Cancer Institute (U.S.) (Cancer Center Support Grant (P30 CA008747).

Estrov and collaborators examine the role of fibrocytes in primary myelofibrosis and propose a novel therapeutic approach., Primary myelofibrosis (PMF) is a fatal neoplastic disease characterized by clonal myeloproliferation and progressive bone marrow (BM) fibrosis thought to be induced by mesenchymal stromal cells stimulated by overproduced growth factors. However, tissue fibrosis in other diseases is associated with monocyte-derived fibrocytes. Therefore, we sought to determine whether fibrocytes play a role in the induction of BM fibrosis in PMF. In this study, we show that BM from patients with PMF harbors an abundance of clonal, neoplastic collagen- and fibronectin-producing fibrocytes. Immunodeficient mice transplanted with myelofibrosis patients’ BM cells developed a lethal myelofibrosis-like phenotype. Treatment of the xenograft mice with the fibrocyte inhibitor serum amyloid P (SAP; pentraxin-2) significantly prolonged survival and slowed the development of BM fibrosis. Collectively, our data suggest that neoplastic fibrocytes contribute to the induction of BM fibrosis in PMF, and inhibiting fibrocyte differentiation with SAP may interfere with this process.

SummaryAlthough clinically tested JAK inhibitors reduce splenomegaly and systemic symptoms, molecular responses are not observed in most myeloproliferative neoplasm (MPN) patients. We previously demonstrated that MPN cells become persistent to type I JAK inhibitors that bind the active conformation of JAK2. We investigated whether CHZ868, a type II JAK inhibitor, would demonstrate activity in JAK inhibitor persistent cells, murine MPN models, and MPN patient samples. JAK2 and MPL mutant cell lines were sensitive to CHZ868, including type I JAK inhibitor persistent cells. CHZ868 showed significant activity in murine MPN models and induced reductions in mutant allele burden not observed with type I JAK inhibitors. These data demonstrate that type II JAK inhibition is a viable therapeutic approach for MPN patients.

Patients with myeloproliferative neoplasms (MPNs) are at significant, cumulative risk of leukemic transformation to acute myeloid leukemia (AML), which is associated with adverse clinical outcome and resistance to standard AML therapies. We performed genomic profiling of post-MPN AML samples; these studies demonstrate somatic tumor protein 53 (TP53) mutations are common in JAK2V617F-mutant, post-MPN AML but not in chronic-phase MPN and lead to clonal dominance of JAK2V617F/TP53-mutant leukemic cells. Consistent with these data, expression of JAK2V617F combined with Tp53 loss led to fully penetrant AML in vivo. JAK2V617F-mutant, Tp53-deficient AML was characterized by an expanded megakaryocyte erythroid progenitor population that was able to propagate the disease in secondary recipients. In vitro studies revealed that post-MPN AML cells were sensitive to decitabine, the JAK1/2 inhibitor ruxolitinib, or the heat shock protein 90 inhibitor 8-(6-iodobenzo[d][1.3]dioxol-5-ylthio)-9-(3-(isopropylamino)propyl)-9H-purine-6-amine (PU-H71). Treatment with ruxolitinib or PU-H71 improved survival of mice engrafted with JAK2V617F-mutant, Tp53-deficient AML, demonstrating therapeutic efficacy for these targeted therapies and providing a rationale for testing these therapies in post-MPN AML.

Somatic mutations in IDH1/IDH2 and TET2 result in impaired TET2-mediated conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The observation that WT1 inactivating mutations anticorrelate with TET2/IDH1/IDH2 mutations in acute myeloid leukemia (AML) led us to hypothesize that WT1 mutations may impact TET2 function. WT1 mutant AML patients have reduced 5hmC levels similar to TET2/IDH1/IDH2 mutant AML. These mutations are characterized by convergent, site-specific alterations in DNA hydroxymethylation, which drive differential gene expression more than alterations in DNA promoter methylation. WT1 overexpression increases global levels of 5hmC, and WT1 silencing reduced 5hmC levels. WT1 physically interacts with TET2 and TET3, and WT1 loss of function results in a similar hematopoietic differentiation phenotype as observed with TET2 deficiency. These data provide a role for WT1 in regulating DNA hydroxymethylation and suggest that TET2 IDH1/IDH2 and WT1 mutations define an AML subtype defined by dysregulated DNA hydroxymethylation., info:eu-repo/semantics/published

The introduction of JAK inhibitors into clinical practice has produced significant clinical benefits for patients with MPNs, including reduction of splenomegaly and improvement in symptom burden. However, JAK inhibitors have demonstrated limited clinical ability to alter the natural history and biology of disease (such as reversal of fibrosis) in MPNs, and have produced only modest reductions in JAK2 V617F allele burden. This has prompted the evaluation of other therapeutic strategies in MPN patients. Heat Shock Protein 90 (Hsp90) inhibitors have recently emerged as promising potential treatment for MPN patients. Hsp90 physically interacts with JAK2, and inhibition of Hsp90 induces degradation of JAK2. Hsp90 is also a client chaperone for a variety of other proteins involved in pathways known to be critical for cancer cell differentiation, proliferation and survival. Preclinical studies have demonstrated that Hsp90 inhibitor therapy results in dose-dependent degradation of JAK2 and inhibition of downstream signaling pathways including STAT5, STAT3 and MAPK, as well as inhibition of growth of cells expressing mutant JAK2. In vivo treatment with Hsp90 inhibitors in MPN retroviral murine models led to normalized of peripheral blood counts, reduction in organomegaly, and improvement in overall survival. Based on these promising preclinical data, we conducted an open label phase II trial to assess the efficacy and confirm the safety of a novel Hsp90 inhibitor, AUY922 (Novartis) in patients with primary myelofibrosis (PMF), post-polycythemia vera and post-essential thrombocythemia myelofibrosis (IPSS-2 or higher) and in patients with polycythemia vera or essential thrombocythemia, who were refractory to, intolerant of, or ineligible for conventional therapy. The primary objective of the study was to determine the efficacy of AUY922 in this patient population. Secondary objectives included confirmation of safety and tolerability along with exploration of how treatment modified the biology of the disease. From 2012-2014, 6 patients were treated, 4 female, median age 55 (53-72), with MPNs; 4 with primary-MF, 1 with PV and 1 with ET, 4 patients were JAK2 V617F positive. Prior treatments included hydroxyurea in 5, anagrelide in 3, interferon in 1, azacitadine and pamidronate in 1. Median length of treatment was 39.5 months (1-145). 3 patients with MF experienced stable disease (including two patients with blast-phase disease), and one experienced an anemia response as measured by the European LeukemiaNet (ELN) response criteria. The patient with ET experienced stable disease as measured by the Revised International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRT). Notable (greater than grade 2) adverse events included diarrhea in 5 patients, night blindness in 4, alkaline phosphatase elevation in 2, nausea in 2, emesis in 2, blurred vision in 1 and CPK elevation in 1 patient Serious Adverse Events (SAEs) included nausea, vomiting, diarrhea and altered mental status. As well, three patients experienced gastrointestinal bleeding as an SAE (grade 2 and 3). Two of these events were associated with an ileocecal ulcer. Two events were deemed to be possibly related to exposure to the study drug. The trial was terminated for this reason. Evaluation of the impact on JAK-STAT signaling is underway with evidence of reduction in total JAK2 protein following infusion (figure 1). Further evaluation of JAK-STAT signaling, impact on cytokine production, and JAK2 allele burden is under evaluation and results will be presented. In summary, treatment with AUY922 in patients with has demonstrated response including stable disease (including in blast-phase myelofibrosis patients) as well as anemia response and clinical improvement in one myelofibrosis patient. These data indicate that Hsp90 inhibition has clinical activity in MPN patients, and that further clinical therapeutic efforts targeting Hsp90 warrant investigation in this patient population. Figure 1. Responses to treatment with AUY922 per Revised International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European LeukemiaNet criteria (ELN). Figure 1. Responses to treatment with AUY922 per Revised International Working Group for Myeloproliferative Neoplasms Research and Treatment (IWG-MRT) and European LeukemiaNet criteria (ELN). Figure 2. Western Blot analysis of total JAK2 from peripheral blood of patient at baseline, cycle 1 day 2, and end of treatment. Figure 2. Western Blot analysis of total JAK2 from peripheral blood of patient at baseline, cycle 1 day 2, and end of treatment. Disclosures Mauro: Pfizer: Consultancy; Novartis Pharmaceutical Corporation: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy; Ariad: Consultancy. Levine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees.

It is unknown if telomere length (TL) is associated with clinical outcome or molecular profile in acute myeloid leukemia (AML). We collected tumor samples from 67 AML patients treated at Memorial Sloan Kettering Cancer Center. DNA extraction was performed using viably frozen peripheral blood and bone marrow mononuclear cells. RainDance was used to amplify all exons of a set of 30 genes commonly mutated in AML. Capture was followed by next-generation sequencing; mutations were called if the variant was supported by >5% of the total number of reads (minimum >10 reads). TL was measured as mean telomere content by qPCR. Patients were assessed for FLT3 and NPM1 mutations, cytogenetics, and outcomes by chart review. Results In our 67 patient AML cohort, median TL was 5.22 kb (range 3.73-8.76). Median age was 64.1 years (range 26.2-84.4). While in healthy individuals TL shortens with age, in our cohort there was no association (R2=0.043). There was no difference in TL between newly diagnosed (ND) and relapsed/refractory (RR) patients or between de novo and secondary AML patients. In the 45 ND patients, there was a trend of early improved survival following sample collection in the longest TL tertile compared to the middle and shortest TL tertiles (86.7% vs. 60.0% and 33.3% at 6 months); however, this association was not statistically significant (p=0.662) (Fig 1). In the 22 RR patients, there was also a trend toward improved OS in the longest TL tertile (60.0% vs. 11.1% and 25.0%, p=0.284). In ND patients, there was no association between TL and primary induction failure or relapse-free survival. Targeted sequencing data for 30 myeloid genes were available in 62 of the 67 patients. Analysis of single mutation correlation with TL showed that patients with IDH1 mutations had significantly longer TL than those without (p=0.02) (Table 1). Moreover, mutations in a set of genes associated with epigenetic functions ( IDH1 / 2 , ASXL1 , DNMT3A , and TET2 ) also correlated with longer TL when examined together as a group (p=0.073) . FLT3 -ITD mutations were associated with shorter TL (p=0.084). The median ages of patients with IDH1 or FLT3 mutations were not different from the rest of the cohort. Of note, FLT3 -mutated patients did have a higher WBC than FLT3 wild-type patients (p

Genomic studies have identified somatic alterations in the majority of myeloproliferative neoplasms (MPN) patients, including JAK2 mutations in the majority of MPN patients and CALR mutations in JAK2-negative MPN patients. However, the role of JAK-STAT pathway activation in different MPNs, and in patients without JAK2 mutations, has not been definitively delineated. We used expression profiling, single nucleotide polymorphism arrays, and mutational profiling to investigate a well-characterized cohort of MPN patients. MPN patients with homozygous JAK2V617F mutations were characterized by a distinctive transcriptional profile. Notably, a transcriptional signature consistent with activated JAK2 signaling is seen in all MPN patients regardless of clinical phenotype or mutational status. In addition, the activated JAK2 signature was present in patients with somatic CALR mutations. Conversely, we identified a gene expression signature of CALR mutations; this signature was significantly enriched in JAK2-mutant MPN patients consistent with a shared mechanism of transformation by JAK2 and CALR mutations. We also identified a transcriptional signature of TET2 mutations in MPN patent samples. Our data indicate that MPN patients, regardless of diagnosis or JAK2 mutational status, are characterized by a distinct gene expression signature with upregulation of JAK-STAT target genes, demonstrating the central importance of the JAK-STAT pathway in MPN pathogenesis.