Passive immunization of macaques with polyclonal anti-SHIV IgG against a heterologous tier 2 SHIV: outcome depends on IgG dose

Background: A key goal for HIV-1 envelope immunogen design is the induction of cross-reactive neutralizing antibodies (nAbs). As AIDS vaccine recipients will not be exposed to strains exactly matching any immunogens due to multiple HIV-1 quasispecies circulating in the human population worldwide, heterologous SHIV challenges are essential for realistic vaccine efficacy testing in primates. We assessed whether polyclonal IgG, isolated from rhesus monkeys (RMs) with high-titer nAbs (termed SHIVIG), could protect RMs against the R5-tropic tier-2 SHIV-2873Nip, which was heterologous to the viruses or HIV-1 envelopes that had elicited SHIVIG. Results: SHIVIG demonstrated binding to HIV Gag, Tat, and Env of different clades and competed with the broadly neutralizing antibodies b12, VRC01, 4E10, and 17b. SHIVIG neutralized tier 1 and tier 2 viruses, including SHIV-2873Nip. NK-cell depletion decreased the neutralizing activity of SHIVIG 20-fold in PBMC assays. Although SHIVIG neutralized SHIV-2873Nip in vitro, this polyclonal IgG preparation failed to prevent acquisition after repeated intrarectal low-dose virus challenges, but at a dose of 400 mg/kg, it significantly lowered peak viremia (P = 0.001). Unexpectedly, single-genome analysis revealed a higher number of transmitted variants at the low dose of 25 mg/kg, implying increased acquisition at low SHIVIG levels. In vitro, SHIVIG demonstrated complement-mediated Ab-dependent enhancement of infection (C’-ADE) at concentrations similar to those observed in plasmas of RMs treated with 25 mg/kg of SHIVIG. Conclusion: Our primate model data suggest a dual role for polyclonal anti-HIV-1 Abs depending on plasma levels upon virus encounter

Characterization of p190-Bcr-Abl chronic myeloid leukemia reveals specific signaling pathways and therapeutic targets

Publisher Copyright: © 2020, The Author(s).The oncogenic protein Bcr-Abl has two major isoforms, p190Bcr-Abl and p210Bcr-Abl. While p210Bcr-Abl is the hallmark of chronic myeloid leukemia (CML), p190Bcr-Abl occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) patients. In CML, p190Bcr-Abl occurs in a minority of patients associating with distinct hematological features and inferior outcomes, yet the pathogenic role of p190Bcr-Abl and potential targeting therapies are largely uncharacterized. We employed next generation sequencing, phospho-proteomic profiling, and drug sensitivity testing to characterize p190Bcr-Abl in CML and hematopoietic progenitor cell line models (Ba/f3 and HPC-LSK). p190Bcr-Abl CML patients demonstrated poor response to imatinib and frequent mutations in epigenetic modifiers genes. In contrast with p210Bcr-Abl, p190Bcr-Abl exhibited specific transcriptional upregulation of interferon, interleukin-1 receptor, and P53 signaling pathways, associated with hyperphosphorylation of relevant signaling molecules including JAK1/STAT1 and PAK1 in addition to Src hyperphosphorylation. Comparable to p190Bcr-Abl CML patients, p190Bcr-Abl cell lines demonstrated similar transcriptional and phospho-signaling signatures. With the drug sensitivity screening we identified targeted drugs with specific activity in p190Bcr-Abl cell lines including IAP-, PAK1-, and Src inhibitors and glucocorticoids. Our results provide novel insights into the mechanisms underlying the distinct features of p190Bcr-Abl CML and promising therapeutic targets for this high-risk patient group.Peer reviewe

Dual-Layer Surface Coating of PLGA-Based Nanoparticles Provides Slow-Release Drug Delivery To Achieve Metronomic Therapy in a Paclitaxel-Resistant Murine Ovarian Cancer Model

Development of drug resistance is a central challenge to the treatment of ovarian cancer. Metronomic chemotherapy decreases the extent of drug-free periods, thereby hindering development of drug resistance. Intraperitoneal chemotherapy allows for treatment of tumors confined within the peritoneum, but achieving sustained tumor-localized chemotherapy remains difficult. We hypothesized that modulating the surface properties of poly­(lactic-<i>co</i>-glycolic acid) (PLGA)-based nanoparticles could enhance their drug retention ability and extend their release profile, thereby enabling metronomic, localized chemotherapy <i>in vivo</i>. Paclitaxel was encapsulated in particles coated with a layer of polydopamine and a subsequent layer of poly­(ethylene glycol) (PEG). These particles achieved a 3.8-fold higher loading content compared to that of nanoparticles formulated from linear PLGA–PEG copolymers. <i>In vitro</i> release kinetic studies and <i>in vivo</i> drug distribution profiles demonstrate sustained release of paclitaxel. Although free drug conferred no survival advantage, low-dose intraperitoneal administration of paclitaxel-laden surface-coated nanoparticles to drug-resistant ovarian tumor-bearing mice resulted in significant survival benefits in the absence of any apparent systemic toxicity

Dual-Layer Surface Coating of PLGA-Based Nanoparticles Provides Slow-Release Drug Delivery To Achieve Metronomic Therapy in a Paclitaxel-Resistant Murine Ovarian Cancer Model

Development of drug resistance is a central challenge to the treatment of ovarian cancer. Metronomic chemotherapy decreases the extent of drug-free periods, thereby hindering development of drug resistance. Intraperitoneal chemotherapy allows for treatment of tumors confined within the peritoneum, but achieving sustained tumor-localized chemotherapy remains difficult. We hypothesized that modulating the surface properties of poly­(lactic-<i>co</i>-glycolic acid) (PLGA)-based nanoparticles could enhance their drug retention ability and extend their release profile, thereby enabling metronomic, localized chemotherapy <i>in vivo</i>. Paclitaxel was encapsulated in particles coated with a layer of polydopamine and a subsequent layer of poly­(ethylene glycol) (PEG). These particles achieved a 3.8-fold higher loading content compared to that of nanoparticles formulated from linear PLGA–PEG copolymers. <i>In vitro</i> release kinetic studies and <i>in vivo</i> drug distribution profiles demonstrate sustained release of paclitaxel. Although free drug conferred no survival advantage, low-dose intraperitoneal administration of paclitaxel-laden surface-coated nanoparticles to drug-resistant ovarian tumor-bearing mice resulted in significant survival benefits in the absence of any apparent systemic toxicity

Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. We have developed synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies were highly selective for STAT3 and bound with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detected no binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, resulted in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 revealed bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibited STAT3-dependent transcriptional activation and disrupted STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms

Selective inhibition of STAT3 signaling using monobodies targeting the coiled-coil and N-terminal domains

The transcription factor STAT3 is frequently activated in human solid and hematological malignancies and remains a challenging therapeutic target with no approved drugs to date. Here, we develop synthetic antibody mimetics, termed monobodies, to interfere with STAT3 signaling. These monobodies are highly selective for STAT3 and bind with nanomolar affinity to the N-terminal and coiled-coil domains. Interactome analysis detects no significant binding to other STATs or additional off-target proteins, confirming their exquisite specificity. Intracellular expression of monobodies fused to VHL, an E3 ubiquitin ligase substrate receptor, results in degradation of endogenous STAT3. The crystal structure of STAT3 in complex with monobody MS3-6 reveals bending of the coiled-coil domain, resulting in diminished DNA binding and nuclear translocation. MS3-6 expression strongly inhibits STAT3-dependent transcriptional activation and disrupts STAT3 interaction with the IL-22 receptor. Therefore, our study establishes innovative tools to interfere with STAT3 signaling by different molecular mechanisms. STAT3 is an attractive therapeutic target but its homology with other STAT proteins complicates the development of selective inhibitors. Here, the authors develop monobodies with high affinity and selectivity for STAT3 and show that they can interfere with cellular STAT3 activity

A robust approach for the generation of functional hematopoietic progenitor cell lines to model leukemic transformation

Studies of molecular mechanisms of hematopoiesis and leukemogenesis are hampered by the unavailability of progenitor cell lines that accurately mimic the situation in vivo. We now report a robust method to generate and maintain LSK (Lin-, Sca-1+, c-Kit+) cells, which closely resemble MPP1 cells. HPCLSKs reconstitute hematopoiesis in lethally irradiated recipient mice over &gt;8 months. Upon transformation with different oncogenes including BCR/ABL, FLT3-ITD, or MLL-AF9, their leukemic counterparts maintain stem cell properties in vitro and recapitulate leukemia formation in vivo. The method to generate HPCLSKs can be applied to transgenic mice, and we illustrate it for CDK6-deficient animals. Upon BCR/ABLp210 transformation, HPCLSKs Cdk6-/- induce disease with a significantly enhanced latency and reduced incidence, showing the importance of CDK6 in leukemia formation. Studies of the CDK6 transcriptome in murine HPCLSK and human BCR/ABL+ cells have verified that certain pathways depend on CDK6 and have uncovered a novel CDK6-dependent signature, suggesting a role for CDK6 in leukemic progenitor cell homing. Loss of CDK6 may thus lead to a defect in homing. The HPCLSK system represents a unique tool for combined in vitro and in vivo studies and enables the production of large quantities of genetically modifiable hematopoietic or leukemic stem/progenitor cells

SBNO2 is a critical mediator of STAT3-driven hematological malignancies

Gain-of-function mutations in the signal transducer and activator of transcription 3 (STAT3) gene are recurrently identified in patients with large granular lymphocytic leu-kemia (LGLL) and in some cases of natural killer (NK)/T-cell and adult T-cell leukemia/ lymphoma. To understand the consequences and molecular mechanisms contributing to disease development and oncogenic transformation, we developed murine hematopoietic stem and progenitor cell models that express mutated STAT3Y640F. These cells show accelerated proliferation and enhanced self-renewal potential. We integrated gene expression analyses and chromatin occupancy profiling of STAT3Y640F-transformed cells with data from patients with T-LGLL. This approach uncovered a conserved set of direct transcriptional targets of STAT3Y640F. Among these, strawberry notch homolog 2 (SBNO2) represents an essential transcriptional target, which was identified by a comparative genome-wide CRISPR/Cas9-based loss-of-function screen. The STAT3-SBNO2 axis is also present in NK-cell leukemia, T-cell non-Hodgkin lymphoma, and NPM-ALK-rearranged T-cell anaplastic large cell lymphoma (T-ALCL), which are driven by STAT3-hyperactivation/ mutation. In patients with NPM-ALK+ T-ALCL, high SBNO2 expression correlates with shorter relapse-free and overall survival. Our findings identify SBNO2 as a potential therapeutic intervention site for STAT3-driven hematopoietic malignancies.Peer reviewe