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Much of what is known about the neurotrophic receptor tyrosine kinase (NTRK) genes in cancer was revealed through identification and characterization of activating Trk fusions across many tumor types. A resurgence of interest in these receptors has emerged owing to the realization that they are promising therapeutic targets. The remarkable efficacy of pan-Trk inhibitors larotrectinib and entrectinib in clinical trials led to their accelerated, tissue-agnostic US Food and Drug Administration (FDA) approval for adult and pediatric patients with Trk-driven solid tumors. Despite our enhanced understanding of Trk biology in solid tumors, the importance of Trk signaling in hematological malignancies is underexplored and warrants further investigation. Herein, we describe mutations in NTRK2 and NTRK3 identified via deep sequencing of 185 patients with hematological malignancies. Ten patients contained a point mutation in NTRK2 or NTRK3; among these, we identified 9 unique point mutations. Of these 9 mutations, 4 were oncogenic (NTRK2A203T, NTRK2R458G, NTRK3E176D, and NTRK3L449F), determined via cytokine-independent cellular assays. Our data demonstrate that these mutations have transformative potential to promote downstream survival signaling and leukemogenesis. Specifically, the 3 mutations located within extracellular (ie, NTRK2A203T and NTRK3E176D) and transmembrane (ie, NTRK3L449F) domains increased receptor dimerization and cell-surface abundance. The fourth mutation, NTRK2R458G, residing in the juxtamembrane domain, activates TrkB via noncanonical mechanisms that may involve altered interactions between the mutant receptor and lipids in the surrounding environment. Importantly, these 4 activating mutations can be clinically targeted using entrectinib. Our findings contribute to ongoing efforts to define the mutational landscape driving hematological malignancies and underscore the utility of FDA-approved Trk inhibitors for patients with aggressive Trk-driven leukemias.

Much of what is known about the neurotrophic receptor tyrosine kinase (NTRK) genes in cancer was revealed through identification and characterization of activating Trk fusions across many tumor types. A resurgence of interest in these receptors has emerged owing to the realization that they are promising therapeutic targets. The remarkable efficacy of pan-Trk inhibitors larotrectinib and entrectinib in clinical trials led to their accelerated, tissue-agnostic US Food and Drug Administration (FDA) approval for adult and pediatric patients with Trk-driven solid tumors. Despite our enhanced understanding of Trk biology in solid tumors, the importance of Trk signaling in hematological malignancies is underexplored and warrants further investigation. Herein, we describe mutations in NTRK2 and NTRK3 identified via deep sequencing of 185 patients with hematological malignancies. Ten patients contained a point mutation in NTRK2 or NTRK3; among these, we identified 9 unique point mutations. Of these 9 mutations, 4 were oncogenic (NTRK2A203T, NTRK2R458G, NTRK3E176D, and NTRK3L449F), determined via cytokine-independent cellular assays. Our data demonstrate that these mutations have transformative potential to promote downstream survival signaling and leukemogenesis. Specifically, the 3 mutations located within extracellular (ie, NTRK2A203Tand NTRK3E176D) and transmembrane (ie, NTRK3L449F) domains increased receptor dimerization and cell-surface abundance. The fourth mutation, NTRK2R458G, residing in the juxtamembrane domain, activates TrkB via noncanonical mechanisms that may involve altered interactions between the mutant receptor and lipids in the surrounding environment. Importantly, these 4 activating mutations can be clinically targeted using entrectinib. Our findings contribute to ongoing efforts to define the mutational landscape driving hematological malignancies and underscore the utility of FDA-approved Trk inhibitors for patients with aggressive Trk-driven leukemias.

AbstractInfectious laryngotracheitis (ILT) is a highly contagious upper respiratory tract disease of chicken caused by a Gallid herpesvirus 1 (GaHV-1) belonging to the genus Iltovirus,and subfamily Alphaherpesvirinaewithin Herpesviridaefamily. The disease is characterized by conjunctivitis, sinusitis, oculo-nasal discharge, respiratory distress, bloody mucus, swollen orbital sinuses, high morbidity, considerable mortality and decreased egg production. It is well established in highly dense poultry producing areas of the world due to characteristic latency and carrier status of the virus. Co-infections with other respiratory pathogens and environmental factors adversely affect the respiratory system and prolong the course of the disease. Latently infected chickens are the primary source of ILT virus (ILTV) outbreaks irrespective of vaccination. Apart from conventional diagnostic methods including isolation and identification of ILTV, serological detection, advanced biotechnological tools such as PCR, quantitative real-time PCR, next generation sequencing, and others are being used in accurate diagnosis and epidemiological studies of ILTV. Vaccination is followed with the use of conventional vaccines including modified live attenuated ILTV vaccines, and advanced recombinant vector vaccines expressing different ILTV glycoproteins, but still these candidates frequently fail to reduce challenge virus shedding. Some herbal components have proved to be beneficial in reducing the severity of the clinical disease. The present review discusses ILT with respect to its current status, virus characteristics, epidemiology, transmission, pathobiology, and advances in diagnosis, vaccination and control strategies to counter this important disease of poultry.

Acute myeloid leukemia is a poor-prognosis cancer commonly stratified by genetic aberrations, but these mutations are often heterogeneous and fail to consistently predict therapeutic response. Here, we combine transcriptomic, proteomic, and phosphoproteomic datasets with ex vivodrug sensitivity data to help understand the underlying pathophysiology of AML beyond mutations. We measure the proteome and phosphoproteome of 210 patients and combine them with genomic and transcriptomic measurements to identify four proteogenomic subtypes that complement existing genetic subtypes. We build a predictor to classify samples into subtypes and map them to a “landscape” that identifies specific drug response patterns. We then build a drug response prediction model to identify drugs that target distinct subtypes and validate our findings on cell lines representing various stages of quizartinib resistance. Our results show how multiomics data together with drug sensitivity data can inform therapy stratification and drug combinations in AML.

NTRK fusions are dominant oncogenic drivers found in rare solid tumors. These fusions have also been identified in more common cancers, such as lung and colorectal carcinomas, albeit at low frequencies. Patients harboring these fusions demonstrate significant clinical response to inhibitors such as entrectinib and larotrectinib. Although current trials have focused entirely on solid tumors, there is evidence supporting the use of these drugs for patients with leukemia. To assess the broader applicability for Trk inhibitors in hematological malignancies, this review describes the current state of knowledge about alterations in the NTRK family in these disorders. We present these findings in relation to the discovery and therapeutic targeting of BCR–ABL1 in chronic myeloid leukemia. The advent of deep sequencing technologies has shown that NTRK fusions and somatic mutations are present in a variety of hematologic malignancies. Efficacy of Trk inhibitors has been demonstrated in NTRK-fusion positive human leukemia cell lines and patient-derived xenograft studies, highlighting the potential clinical utility of these inhibitors for a subset of leukemia patients.

IMPORTANCE: Surrogate end points in oncology trade the advantage of reducing the time needed to conduct clinical trials for the disadvantage of greater uncertainty regarding the treatment effect on patient-centered end points, such as overall survival (OS) and quality of life. OBJECTIVE: To quantify the amount of time saved through the acceptance of surrogate end points, including response rate (RR) and progression-free survival (PFS). DESIGN, SETTING, AND PARTICIPANTS: This retrospective study of US Food and Drug Administration (FDA) oncology approvals and their drug registration trials based on actual publication analyzed the original and updated clinical trials data that led to FDA-approved drug indications in oncology from 2006 to 2017 by using existing publications, conference abstracts, and package inserts from the FDA. Data related to cancer type, line of therapy (first-line, second-line, and third- or later-line treatment of advanced or metastatic disease), FDA approval type, end point basis for approval (RR, PFS, or OS/quality of life), sample size, accrual rate, and drug RR were extracted by March 23, 2018. All data were analyzed by July 13, 2018. MAIN OUTCOMES AND MEASURES: The main outcome was the study duration needed to complete the primary end point analysis used for each drug indication approval. This was estimated from reported enrollment dates, analysis cutoff dates, time to response, median duration of response, median PFS, and median OS. RESULTS: In total, 188 distinct indications among 107 cancer drugs were identified. The RR was more often used for FDA approval in subsequent lines of therapy (17 of 71 drug indications [24%] in first-line therapy vs 34 of 77 drug indications [44%] in second-line therapy vs 19 of 24 drug indications [79%] in third- or later-line therapy, P < .001). Study duration for PFS (median, 31 [range, 10-104] months) was similar to that for OS (median, 33 [range, 12-117] months; P = .31), whereas study duration for RR (median, 25 [range, 11-54] months) was shorter than that for OS (P = .001). In multivariate analysis, compared with using OS, use of PFS as the end point was associated with study durations that were shorter by a mean of 11 months (95% CI, 5-17 months), and the use of RR as the end point was associated with study durations that were shorter by a mean of 19 months (95% CI, 13-25 months). CONCLUSIONS AND RELEVANCE: From the findings of this study, an estimated 11 months appeared to be needed (ie, approximately 12% longer in the drug development cycle) to assess the OS benefit of a cancer drug. This study’s findings suggest that this must be weighed against the downside of increased uncertainty of clinical benefit arising from using surrogate end points.

Introduction :NCAM1 (CD56) is a cellular adhesion molecule highly expressed on natural killer (NK) cells and other immune cells. NCAM1 is aberrantly expressed in 20% of patients with acute myeloid leukemia (AML) and is associated with resistance to chemotherapy, upregulation of the MAPK pathway, and poor prognosis. The significance of NCAM1 in the setting of venetoclax (VEN) therapy has not been explored. We set out to determine the functional and prognostic role of CD56 in vitroand in newly diagnosed (ND) patients receiving VEN-based therapy.

Background: In many cancers, predictive factors are important for recognition of high-risk patients and amongst individualizing treatment. The understanding of these mechanisms might provide novel and useful approaches for preventing, diagnosing and treating different cancers. Objective: The main objective of this review is to extend the current knowledge on the various tumor biomarkers targeting prognosis and therapies of human cancers. Methods: The present review is based on the extensive churning, analyzing and compilation of the salient information on tumor biomarkers from the authentic published literature available in PubMed and other scientific databases. The information also includes the implicative role of nucleic acids, apolipoproteins, inflammatory biomolecules, receptors, DNA modification, carbohydrate antigens and metabolite signatures as biomarkers for cancer. Results: In this review, we have summarized some tumor biomarkers, which would improve prognostic efficiency and accuracy among patients with cancer particularly for ovarian, lung, breast, melanoma and pancreatic cancer. Conclusion: This review provides in-depth insights of the use and importance of cancer biomarkers in our understanding as well improve knowledge regarding cancer management in clinical practice that will facilitate a more effective prognosis with least undesired systemic toxicity. However, development and evaluation of cancer biomarkers demand a complete understanding of the molecular processes and cellular mechanisms during the onset of cancer; as well how a little modification in regulatory metabolites, proteins or genes can disrupt various kinds of cellular functions and lead to cancer.

Repair of peripheral nerve defects with current synthetic, tubular nerve conduits generally shows inferior recovery when compared with using nerve autografts, the current gold standard. We tested the ability of composite collagen and hyaluronan hydrogels, with and without the nerve growth factor (NGF), to stimulate neurite extension on a promising aligned, nanofiber poly-L-lactide-co-caprolactone (PLCL) scaffold. In vitro, the hydrogels significantly increased neurite extension from dorsal root ganglia explants. Consistent with these results, the addition of hydrogels as luminal fillers within aligned, nanofiber tubular PLCL conduits led to improved sensory function compared to autograft repair in a critical-size defect in the sciatic nerve in a rat model. Sensory recovery was assessed 3 and 12 weeks after repair using a withdrawal assay from thermal stimulation. The addition of hydrogel did not enhance recovery of motor function in the rat model. The NGF led to dose-dependent improvements in neurite out-growth in vitro, but did not have a significant effect in vivo. In summary, composite collagen/hyaluronan hydrogels enhanced sensory neurite outgrowth in vitroand sensory recovery in vivo. The use of such hydrogels as luminal fillers for tubular nerve conduits may therefore be useful in assisting restoration of protective sensation following peripheral nerve injury.

CD1d expression by dendritic cells is dispensable for NKT cell‐enhanced antibody responses, but is required for NKT cell‐enhanced CD8+ T cell responses. CD1d‐restricted type I NKT cells provide help for specific antibody production. B cells, which have captured and presented a T‐dependent, antigen‐derived peptide on MHC class II and CD1d‐binding glycolipid α‐GC on CD1d, respectively, activate Th and NKT cells to elicit B cell help. However, the role of the DC CD1d in humoral immunity remains unknown. We therefore constructed mixed bone marrow chimeras containing CD1d‐expressing, DTR‐transgenic DCs and CD1d+or CD1d−nontransgenic DCs. Following DT‐mediated DC ablation and immunization, we observed that the primary and secondary antibody responses were equivalent in the presence of CD1d+and CD1d−DCs. In contrast, a total ablation of DCs delayed the primary antibody response. Further experiments revealed that depletion of CD1d+DCs blocked in vivo expansion of antigen‐specific cytotoxic (CD8+) T lymphocytes. These results provide a clear demonstration that although CD1d expression on DCs is essential for NKT‐enhanced CD8+T cell expansion, it is dispensable for specific antibody production.

ABSTRACTThe current Bacillus anthracisvaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracisbut has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (α-galactosylceramide [α-GC]) at the time of immunization improves PA-specific Ab responses. We observed that α-GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d−/−mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In Jα281−/−mice expressing CD1d but lacking type I α-GC-reactive NKT cells, α-GC did not enhance the Ab response. In vitroneutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivoprotection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracisthat increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

The current Bacillus anthracis vaccine consists largely of protective antigen (PA), the protein of anthrax toxin that mediates entry of edema factor (EF) or lethal factor (LF) into cells. PA induces protective antibody (Ab)-mediated immunity against Bacillus anthracis but has limited efficacy and duration. We previously demonstrated that activation of CD1d-restricted natural killer-like T cells (NKT) with a CD1d-binding glycolipid led to enhanced Ab titers specific for foreign antigen (Ag). We therefore tested the hypothesis that activation of NKT cells with the CD1d ligand (-galactosylceramide [-GC]) at the time of immunization improves PA-specific Ab responses. We observed that -GC enhanced PA-specific Ab titers in C57BL/6 mice. In CD1d–/–mice deficient in type I and type II NKT cells the anti-PA Ab response was diminished. In J281–/–mice expressing CD1d but lacking type I -GC-reactive NKT cells, -GC did not enhance the Ab response. In vitro neutralization assays were performed and showed that the Ab titers correlated with protection of macrophages against anthrax lethal toxin (LT). The neutralization capacity of the Ab was further tested in lethal challenge studies, which revealed that NKT activation leads to enhanced in vivo protection against LT. Anti-PA Ab titers, neutralization, and protection were then measured over a period of several months, and this revealed that NKT activation leads to a sustained protective Ab response. These results suggest that NKT-activating CD1d ligands could be exploited for the development of improved vaccines for Bacillus anthracis that increase not only neutralizing Ab titers but also the duration of the protection afforded by Ab.

Tyner: Incyte: Research Funding; Janssen: Research Funding; Syros: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Agios: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Aptose: Research Funding; Array: Research Funding; Genentech: Research Funding; Constellation: Research Funding; Takeda: Research Funding. Druker:Henry Stewart Talks: Patents & Royalties; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; McGraw Hill: Patents & Royalties; MolecularMD (acquired by ICON): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Millipore (formerly Upstate Biotechnology): Patents & Royalties; Merck & Co: Patents & Royalties; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Dana-Farber Cancer Institute: Patents & Royalties; EnLiven: Consultancy, Research Funding; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics Inc. (formerly Lorus): Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Blueprint Medicines: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Oregon Health & Science University: Patents & Royalties; Patient True Talks: Consultancy; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics (formerly Beta Cat Pharmaceuticals): Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Research Funding. Traer:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Notable Labs: Consultancy, Current equity holder in private company; Astellas: Membership on an entity's Board of Directors or advisory committees.

Activating mutations in the tyrosine kinase receptor FLT3 are observed in ~35% of all acute myeloid leukemia (AML) cases. Multiple FLT3 inhibitors are currently in clinical development and while most patients initially respond well to FLT3 inhibition, resistance inevitably develops in a period of months. During the initial response to FLT3 inhibitors, residual leukemia cells are able to survive and persist in the marrow microenvironment, which facilitates early resistance. Over time, leukemia cells develop intrinsic mechanisms of resistance, that are not dependent upon the microenvironment, which leads to late resistance and disease relapse.

Homing of mast cell progenitors (MCps) to the mouse small intestine involves the interaction of α4β7 integrin with mucosal addressin cellular adhesion molecule-1 (MAdCAM-1). We now demonstrate the dependence of this process on CXC chemokine receptor 2 (CXCR2) and vascular cell adhesion molecule-1 (VCAM-1) using null strains and mice sublethally irradiated and bone marrow (BM) reconstituted (SIBR) with wild-type or null BM or with wild-type BM followed by administration of blocking antibody. The intestinal MCp concentration in CXCR2-/- mice was reduced by 67%, but was unaltered in CC chemokine receptor 2-/- (CCR2-/-), CCR3-/-, or CCR5-/- mice. SIBR mice given CXCR2-/- BM had an intestinal MCp concentration that was 76% less than that in BALB/c BM reconstituted mice. Antibody blockade of VCAM-1 or of CXCR2 in SIBR mice reduced intestinal MCp reconstitution, and mice lacking endothelial VCAM-1 also had a marked reduction relative to wild-type mice. Finally, the half-life of intestinal MCps in wild-type mice was less than one week on the basis of a more than 50% reduction by administration of anti-α4β7 integrin or anti-CXCR2. Thus, the establishment and maintenance of MCps in the small intestine is a dynamic process that requires expression of the α4β7 integrin and the α-chemokine receptor CXCR2.

We generated vascular cell adhesion molecule (VCAM)-1 “knock-in” mice and Cre recombinase transgenic mice to delete the VCAM-1 gene (vcam-1) in whole mice, thereby overcoming the embryonic lethality seen with conventional vcam-1–deficient mice. vcam-1 knock-in mice expressed normal levels of VCAM-1 but showed loss of VCAM-1 on endothelial and hematopoietic cells when interbred with a “TIE2Cre” transgene. Analysis of peripheral blood from conditional vcam-1–deficient mice revealed mild leukocytosis, including elevated immature B cell numbers. Conversely, the bone marrow (BM) had reduced immature B cell numbers, but normal numbers of pro-B cells. vcam-1–deficient mice also had reduced mature IgD+ B and T cells in BM and a greatly reduced capacity to support short-term migration of transferred B cells, CD4+ T cells, CD8+ T cells, and preactivated CD4+ T cells to the BM. Thus, we report an until now unappreciated dominant role for VCAM-1 in lymphocyte homing to BM.

ABSTRACTLiver-stage antigen 1 (LSA-1) is a potential vaccine candidate against preerythrocytic stages of malaria. We report here the immunogenicity of linear synthetic constructs delineated as TH-cell determinants from the nonrepeat regions ofPlasmodium falciparumLSA-1 in murine models and human subjects from areas where malaria is endemic in Rajasthan State, India. Seven peptide constructs (LS1.1 to LS1.7) corresponding to predicted T-cell sites from both the N- and C-terminal regions and peptide LS1R from a repeat region of PfLSA-1 were synthesized to analyze the cellular immune responses. These linear peptides were also tested for humoral responses in order to determine if there were any overlapping B-cell epitopes in the predicted T-cell sites. Most peptides induced cellular responses in peptide-immunized BALB/c and C57BL/6 mice as measured by proliferation and cytokine analysis. Cross-reactive T-cell recognition of P. falciparum-based peptides in Plasmodium berghei-immune animals was evaluated, but only one peptide, LS1.2 (amino acids 1742 to 1760) triggered T-cell proliferation and interleukin-2 and gamma interferon secretion in P. berghei-immune splenocytes of BALB/c and C57BL/6 mice as well as in Thamnomys gazellae(natural host of P. bergheiANKA). In an enzyme-linked immunosorbent assay with the peptides, only one peptide, LS1.1, was recognized by anti-P. bergheiliver-stage serum. Three peptides (LS1.1, LS1.2, and LS1.3) of the eight peptides tested in this study were recognized by a relatively large percentage ofP. falciparum-exposed human subjects; the reactivities ranged from ∼45% for LS1.3 to ∼60% for LS1.1 and LS1.2. Interestingly, all of the eight putative T-cell determinants were also recognized by the sera collected from malaria patients, although the response was variable in nature. These TH- and B-cell epitopes may be of potential value for preerythrocytic antigen-based malaria subunit vaccine formulations.

Background:FMS-like tyrosine kinase (FLT3) is one the most frequently mutated genes in AML and is associated with poor prognosis. FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations occur in up to 30% and 5-10% of AML, respectively. Several small molecule FLT3 inhibitors (FLT3i) have been developed but their use as single agents is limited due to the development of drug resistance. Our lab developed a two-step model of early and late resistance to FLT3i that recapitulates resistance in AML patients (Traer et al. Cancer Res. 2016; Joshi et al. Cancer Cell 2021). Early resistance, also known as AML persistence, is the stage when residual AML cells are dependent upon the marrow microenvironment for survival and patients are clinically responding. Late resistance to GILT was characterized by expansion of intrinsic mutations, with NRAS mutations being the most frequent mutation, in addition to a few gatekeeper FLT3 mutations. Current therapies are looking at combinations to overcome GILT resistance, including chemotherapy, hypomethylating agents (HMAs), and venetoclax (VEN) +/- HMAs. GILT+VEN, in particular, has shown good initial activity in relapsed/refractory FLT3 AML patients (Daver et al. ASH 2020), however the mechanism of resistance to this combination is unknown.

Tyner: Seattle Genetics: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Agios: Research Funding; Schrodinger: Research Funding. Traer: ImmunoGen: Membership on an entity's Board of Directors or advisory committees; Schrodinger: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Servier/Agios: Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees.

ABSTRACTA cysteine-containing peptide motif, EWSPCSVTCG, is found highly conserved in the circumsporozoite protein (CSP) and the thrombospondin-related anonymous protein (TRAP) of all thePlasmodiumspecies analyzed so far and has been shown to be crucially involved in the sporozoite invasion of hepatocytes. We have recently shown that peptide sequences containing this motif, and also the antibodies raised against the motif, inhibit the merozoite invasion of erythrocytes. However, during natural infection, and upon immunization with recombinant CSP, this motif represents a cryptic epitope. Here we present the results of immunization studies with two linear multiepitopic constructs, a 60-residue (P60) and a 32-residue (P32) peptide, containing the conserved motif sequence. Both the peptides per se generated high levels of specific antibodies in BALB/c mice. P32 was found to be genetically restricted toH-2dand H-2bhaplotypes of mice, whereas P60 was found to be immunogenic in five different strains of mice. The antibody response was predominantly targeted to the otherwise cryptic, conserved motif sequence in P60. Anti-P60 antibodies specifically stained the asexual blood stages ofPlasmodium falciparumand Plasmodium yoeliiin an immunofluorescence assay, recognized a 60- to 65-kDa parasite protein in an immunoblot assay, and blocked P. falciparummerozoite invasion of erythrocytes in a dose-dependent manner. Immunization with P60 also induced significant levels of the cytokines interleukin-2 (IL-2), IL-4, and gamma interferon in BALB/c mice. Moreover, >60% of mice immunized with P60 survived a heterologous challenge infection with a lethal strain of P. yoelii. These results indicate that appropriate medium-sized synthetic peptides might prove useful in generating specific immune responses to an otherwise cryptic but critical and putatively protective epitope in an antigen and could form part of a multicomponent malaria vaccine.

Tyner: Constellation: Research Funding; Incyte: Research Funding; Agios: Research Funding; Gilead: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Petra: Research Funding; AstraZeneca: Research Funding; Janssen: Research Funding; Genentech: Research Funding; Takeda: Research Funding; Syros: Research Funding; Aptose: Research Funding; Array: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Aptose: Research Funding; Array: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; AstraZeneca: Research Funding; Constellation: Research Funding; Syros: Research Funding; Takeda: Research Funding; Agios: Research Funding; Genentech: Research Funding. Druker:ICON: Other: Scientific Founder of Molecular MD, which was acquired by ICON in Feb. 2019; Bristol-Myers Squibb: Patents & Royalties, Research Funding; GRAIL: Equity Ownership, Other: former member of Scientific Advisory Board; Patient True Talk: Consultancy; Gilead Sciences: Other: former member of Scientific Advisory Board; Monojul: Other: former consultant; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Beat AML LLC: Other: Service on joint steering committee; OHSU (licensing fees): Patents & Royalties: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees ; Celgene: Consultancy; ALLCRON: Membership on an entity's Board of Directors or advisory committees; Amgen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Beta Cat: Membership on an entity's Board of Directors or advisory committees, Other: Stock options; Novartis: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Patents & Royalties: Patent 6958335, Treatment of Gastrointestinal Stromal Tumors, exclusively licensed to Novartis, Research Funding; Bristol-Myers Squibb: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Pfizer: Other: PI or co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; Merck & Co: Patents & Royalties: Dana-Farber Cancer Institute license #2063, Monoclonal antiphosphotyrosine antibody 4G10, exclusive commercial license to Merck & Co; Dana-Farber Cancer Institute (antibody royalty): Patents & Royalties: #2524, antibody royalty; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Honoraria; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CureOne: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Aileron Therapeutics: #2573, Constructs and cell lines harboring various mutations in TNK2 and PTPN11, licensing fees , Membership on an entity's Board of Directors or advisory committees.

The neurotrophic receptor tyrosine kinases (NTRKs) are a family of genes-NTRK1, NTRK2, and NTRK3-that encode TrkA, TrkB, and TrkC receptors, respectively. These surface receptors consist of an extracellular domain for ligand binding, a single-pass transmembrane domain, and an intracellular domain with kinase activity. Upon ligand binding, these receptors homodimerize, which in turn leads to trans-phosphorylation of key tyrosine residues in the intracellular domain that further activate several downstream pathways including JAK/STAT, PI3K/AKT, and RAS/MAPK to promote proliferation, differentiation, and survival.

HIV latency may not be governed by a lack of transcriptional initiation, but rather by blocks to RNA elongation, polyadenylation, and splicing.

Broyles: EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Curtis:EpimedX, LLC: Employment, Equity Ownership, Research Funding. Roth:EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Floyd:EpimedX, LLC: Employment, Equity Ownership, Research Funding. Belegu:EpimedX, LLC: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Floyd:EpimedX, LLC: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding.

To test the phenotypic cure we have discovered for SCD and malaria in vivo in mice and in vitro in human red blood cell precursors from SCD patients in two-phase cultures.Gene regulation of developmental hemoglobin switching provides phenotypic cures for beta thalassemias, sickle cell disease, and malaria since it is known that reactivation of fetal globin expression alleviates all these disorders. We discovered a protein that regulates this developmental switch. Ferritin heavy chain (FtH) represses adult β-globin and activates γ(fetal)-globin gene expression in embryonic/K562 erythroid cells (Broyles et al., PNAS98: 9145, 2001; US Patents #7,517,669, #7,718,669, & #2009/0232783 A1; EU Patent # EP1354032B1; Australia patent #2002217964), leading us to propose FtH as a therapeutic agent.Normal C57BL/6 mice, transgenics carrying the complete human beta-globin gene cluster (Beta-YAC Tg), and transgenic mice that express human FtH in definitive erythroid cells have been used under an IACUC-approved protocol to show the safety and efficacy of human FtH and our phytotherapeutic in vivo. Erythroid precursor cells from pediatric SCD patients obtained under an IRB-approved protocol and cultured in a two-phase system allow direct testing of an FtH expression plasmid, FtH protein, and our phytotherapeutic for manipulating Hb phenotypes. Hemoglobins were identified and quantified by HPLC; fetal Hb was confirmed by immunofluorescence.Human FtH transgenic mice, in which the FtH gene is driven by a truncated b-promoter lacking the FtH-binding motif, express human FtH in definitive erythroid cells which results in repression of bMajor-globin but not bMinor-globin. Thus, these TgFtH mice are born with a reduced ratio of bMajor/bMinor globins, resulting in α-globin chain excess and a mild b-thalassemia. In applying this therapy to humans, thalassemia would not be expected since FtH also activates g (fetal)-globin, preventing the chain imbalance. Recent results with normal mice and Beta-YacTg mice show that FtH protein and our phytotherapeutic are both well tolerated in vivo. We have used numbers of target cells (found in the FtH transgenics) as an initial screen for determining an effective dosing regimens. Initial results indicate that both therapeutics are altering the Hb phenotype as predicted.With cultured erythroid precursor cells from the pediatric SCD patients, our results show a complete switch from HbS to HbF with each mode of delivery of FtH or the phytotherapeutic. These results were replicated 15 times using cells from 9 SCD donors, as well as in erythroid precursors from normal donors. Human erythroid precursor cells have surface receptors specific for FtH. Immunofluorescence showed that human FtH is taken into the precursor cells in culture; and confocal microscopy confirmed nuclear localization of exogenously applied FtH. These results suggest that the purified protein and/or the phytotherapeutic can be directly delivered without gene therapy. Therefore, these methods of generating a phenotypic cure for malaria, beta-thal, and SCD should be inexpensive to deliver in vivo. Supported in part by donations to The Sickle Cell Cure Foundation and by a Grand Challenges in Global Health grant from the Bill & Melinda Gates Foundation.No relevant conflicts of interest to declare.

Abstract 903