The SARS-CoV-2 origin dilemma: Zoonotic transfer or laboratory leak?

The COVID-19 pandemic is responsible for millions of deaths worldwide yet its origin remains unclear. Two potential scenarios of how infection of humans initially occurred include zoonotic transfer from wild animals and a leak of the pathogen from a research laboratory. The Wuhan wet markets where wild animals are sold represent a strong scenario for zoonotic transfer. However, isolation of SARS-CoV-2 or its immediate predecessor from wild animals in their natural environment has yet to be documented. Due to incomplete evidence for a zoonotic origin, a laboratory origin is plausible. The Wuhan Institute of Virology is at the epicenter of the pandemic and their work has included manipulation of wild-type coronavirus to enable infection of human cells. Although stronger evidence supports the zoonotic transfer, inconclusive reports maintain the laboratory leak hypothesis alive. It is imperative to reach a factual conclusion to prevent future pandemics.

Structural Analysis of Janus Tyrosine Kinase Variants in Hematological Malignancies: Implications for Drug Development and Opportunities for Novel Therapeutic Strategies.

Janus tyrosine kinase (JAK) variants are known drivers for hematological disorders. With the full-length structure of mouse JAK1 being recently resolved, new observations on the localization of variants within closed, open, and dimerized JAK structures are possible. Full-length homology models of human wild-type JAK family members were developed using the Glassman et al. reported mouse JAK1 containing the V658F structure as a template. Many mutational sites related to proliferative hematological disorders reside in the JH2 pseudokinase domains facing the region important in dimerization of JAKs in both closed and open states. More than half of all JAK gain of function (GoF) variants are changes in polarity, while only 1.2% are associated with a change in charge. Within a JAK1-JAK3 homodimer model, IFNLR1 (PDB ID7T6F) and the IL-2 common gamma chain subunit (IL2Rγc) were aligned with the respective dimer implementing SWISS-MODEL coupled with ChimeraX. JAK3 variants were observed to encircle the catalytic site of the kinase domain, while mutations in the pseudokinase domain align along the JAK-JAK dimerization axis. FERM domains of JAK1 and JAK3 are identified as a hot spot for hematologic malignancies. Herein, we propose new allosteric surfaces for targeting hyperactive JAK dimers.

JAK1 Pseudokinase V666G Mutant Dominantly Impairs JAK3 Phosphorylation and IL-2 Signaling.

Overactive Janus kinases (JAKs) are known to drive leukemia, making them well-suited targets for treatment. We sought to identify new JAK-activating mutations and instead found a JAK1-inactivating pseudokinase mutation, V666G. In contrast to other pseudokinase mutations that canonically lead to an active kinase, the JAK1 V666G mutation led to under-activation seen by reduced phosphorylation. To understand the functional role of JAK1 V666G in modifying kinase activity we investigated its influence on other JAK kinases and within the Interleukin-2 pathway. JAK1 V666G not only inhibited its own activity, but its presence could inhibit other JAK kinases. These findings provide new insights into the potential of JAK1 pseudokinase to modulate its own activity, as well as of other JAK kinases. Thus, the features of the JAK1 V666 region in modifying JAK kinases can be exploited to allosterically inhibit overactive JAKs.

Quadruple and Truncated MEK3 Mutants Identified from Acute Lymphoblastic Leukemia Promote Degradation and Enhance Proliferation.

Compared to other ethnicities, Hispanic children incur the highest rates of leukemia, and most cases are diagnosed as Acute Lymphoblastic Leukemia (ALL). Despite improved treatment and survival for ALL, disproportionate health outcomes in Hispanics persist. Thus, it is essential to identify oncogenic mutations within this demographic to aid in the development of new strategies to diagnose and treat ALL. Using whole-exome sequencing, five single nucleotide polymorphisms within mitogen-activated protein kinase 3 (MAP2K3) were identified in an ALL cancer patient library from the U.S./Mexico border. MAP2K3 R26T and P11T are located near the substrate-binding site, while R65L and R67W localized to the kinase domain. Truncated-MAP2K3 mutant Q73* was also identified. Transfection in HEK293 cells showed that the quadruple-MEK3 mutant (4M-MEK3) impacted protein stability, inducing degradation and reducing expression. The expression of 4M-MEK3 could be rescued by cysteine/serine protease inhibition, and proteasomal degradation of truncated-MEK3 occurred in a ubiquitin-independent manner. MEK3 mutants displayed reduced auto-phosphorylation and enzymatic activity, as seen by decreases in p38 phosphorylation. Furthermore, uncoupling of the MEK3/p38 signaling pathway resulted in less suppressive activity on HEK293 cell viability. Thus, disruption of MEK3 activation may promote proliferative signals in ALL. These findings suggest that MEK3 represents a potential therapeutic target for treating ALL.

Bif-1 Interacts with Prohibitin-2 to Regulate Mitochondrial Inner Membrane during Cell Stress and Apoptosis.

BACKGROUND: Mitochondria are dynamic organelles that undergo fission and fusion. During cell stress, mitochondrial dynamics shift to fission, leading to mitochondrial fragmentation, membrane leakage, and apoptosis. Mitochondrial fragmentation requires the cleavage of both outer and inner membranes, but the mechanism of inner membrane cleavage is unclear. Bif-1 and prohibitin-2 may regulate mitochondrial dynamics. METHODS: We used azide-induced ATP depletion to incite cell stress in mouse embryonic fibroblasts and renal proximal tubular cells, and renal ischemia-reperfusion to induce stress in mice. We also used knockout cells and mice to determine the role of Bif-1, and used multiple techniques to analyze the molecular interaction between Bif-1 and prohibitin-2. RESULTS: Upon cell stress, Bif-1 translocated to mitochondria to bind prohibitin-2, resulting in the disruption of prohibitin complex and proteolytic inactivation of the inner membrane fusion protein OPA1. Bif-1-deficiency inhibited prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis. Domain deletion analysis indicated that Bif-1 interacted with prohibitin-2 via its C-terminus. Notably, mutation of Bif-1 at its C-terminal tryptophan-344 not only prevented Bif-1/prohibitin-2 interaction but also reduced prohibitin complex disruption, OPA1 proteolysis, mitochondrial fragmentation, and apoptosis, supporting a pathogenic role of Bif-1/prohibitin-2 interaction. In mice, Bif-1 bound prohibitin-2 during renal ischemia/reperfusion injury, and Bif-1-deficiency protected against OPA1 proteolysis, mitochondrial fragmentation, apoptosis and kidney injury. CONCLUSIONS: These findings suggest that during cell stress, Bif-1 regulates mitochondrial inner membrane by interacting with prohibitin-2 to disrupt prohibitin complexes and induce OPA1 proteolysis and inactivation.

Isoproterenol-induced beta-2 adrenergic receptor activation negatively regulates interleukin-2 signaling.

Regulation of intracellular signaling pathways in lymphocytes is critical for cell homeostasis and immune response. Interleukin-2 (IL-2), a key regulator of lymphocytes, signals following receptor-ligand engagement and subsequent recruitment and activation of effector proteins including JAKs and STATs. Lymphocytes can also be regulated by the central nervous system through the ß2 adrenergic receptor (ß2AR) pathway which can affect cell trafficking, proliferation, differentiation, and cytokine production. The cross-talk between these two signaling pathways represents an important mechanism that has yet to be fully elucidated. The present study provides evidence for communication between the IL-2 receptor (IL-2R) and ß2AR. Treatment of human lymphoid cell lines with the ß2AR agonist isoproterenol (ISO) alone increased cAMP levels and mediated a stimulatory response by activating AKT and ERK to promote cell viability. Interestingly, ISO activation of ß2AR also induced threonine phosphorylation of the IL-2Rß. In contrast, ISO treatment prior to IL-2 stimulation produced an inhibitory signal that disrupted IL-2 induced activation of the JAK/STAT, MEK/ERK, and PI3K pathways by inhibiting the formation of the IL-2R beta-gamma chain complex, and subsequently cell proliferation. Moreover, γc-family cytokines-mediated STAT5 activation was also inhibited by ISO. These results suggest a molecular mechanism by which ß2AR signaling can both stimulate and suppress lymphocyte responses and thus explain how certain therapeutic agents, such as vasodilators, may impact immune responsiveness.

Exploration of Nanoparticle-Mediated Photothermal Effect of TMB-H2O2 Colorimetric System and Its Application in a Visual Quantitative Photothermal Immunoassay.

The exploration of new physical and chemical properties of materials and their innovative application in different fields are of great importance to advance analytical chemistry, material science, and other important fields. Herein, we, for the first time, discovered the photothermal effect of an iron oxide nanoparticles (NPs)-mediated TMB (3,3',5,5'-tetramethylbenzidine)-H2O2 colorimetric system, and applied it toward the development of a new NP-mediated photothermal immunoassay platform for visual quantitative biomolecule detection using a thermometer as the signal reader. Using a sandwich-type proof-of-concept immunoassay, we found that the charge transfer complex of the iron oxide NPs-mediated one-electron oxidation product of TMB (oxidized TMB) exhibited not only color changes, but also a strong near-infrared (NIR) laser-driven photothermal effect. Hence, oxidized TMB was explored as a new sensitive photothermal probe to convert the immunoassay signal into heat through the near-infrared laser-driven photothermal effect, enabling simple photothermal immunoassay using a thermometer. Based on the new iron oxide NPs-mediated TMB-H2O2 photothermal immunoassay platform, prostate-specific antigen (PSA) as a model biomarker can be detected at a concentration as low as 1.0 ng·mL-1 in normal human serum. The discovered photothermal effect of the colorimetric system and the developed new photothermal immunoassay platform open up a new horizon for affordable detection of disease biomarkers and have great potential for other important material and biomedical applications of interest.

Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth.

BACKGROUND: Although there are reports that metronomic cyclophosphamide (CTX) can be immune stimulating, the impact of its combination with anti-CTLA-4 immunotherapy for the treatment of cancer remains to be evaluated. METHODS: Murine EMT-6/P breast cancer, or its cisplatin or CTX-resistant variants, or CT-26 colon, were implanted into Balb/c mice. Established tumours were monitored for relative growth following treatment with anti-CTLA-4 antibody alone or in combination with; (a) metronomic CTX (ldCTX; 20 mg kg-1 day-1), b) bolus (150 mg kg-1) plus ldCTX, or (c) sequential treatment with gemcitabine (160 mg kg-1 every 3 days). RESULTS: EMT-6/P tumours responded to anti-CTLA-4 therapy, but this response was less effective when combined with bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with regimens of either sequential or concomitant gemcitabine, including in orthotopic EMT-6 tumours, and independently of the schedule of drug administration. Tumour responses were confirmed with CT-26 tumours but were less pronounced in drug-resistant EMT-6/CTX or EMT-6/DDP tumour models than in the parent tumour. A number of tumour bearing mice developed spontaneous metastases under continuous therapy. The majority of cured mice rejected tumour re-challenges. CONCLUSIONS: Metronomic CTX can be combined with anti-CTLA-4 therapy, but this therapy is impaired by concomitant bolus CTX. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in chemotherapy-naive tumours, although tumour relapses can occur, in some cases accompanied by the development of spontaneous metastases.

Interleukin-2 Receptor ß Thr-450 Phosphorylation Is a Positive Regulator for Receptor Complex Stability and Activation of Signaling Molecules.

T, B, and natural killer cells are required for normal immune response and are regulated by cytokines such as IL-2. These cell signals are propagated following receptor-ligand engagement, controlling recruitment and activation of effector proteins. The IL-2 receptor ß subunit (IL-2Rß) serves in this capacity and is known to be phosphorylated. Tyrosine phosphorylation of the ß chain has been studied extensively. However, the identification and putative regulatory roles for serine and threonine phosphorylation sites have yet to be fully characterized. Using LC-MS/MS and phosphospecific antibodies, a novel IL-2/IL-15 inducible IL-2Rß phosphorylation site (Thr-450) was identified. IL-2 phosphokinetic analysis revealed that phosphorylation of IL-2Rß Thr-450 is rapid (2.5 min), transient (peaks at 15 min), and protracted compared with receptor tyrosine phosphorylation and occurs in multiple cell types, including primary human lymphocytes. Pharmacological and siRNA-mediated inhibition of various serine/threonine kinases revealed ERK1/2 as a positive regulator, whereas purified protein phosphatase 1 (PP1), dephosphorylated Thr-450 in vitro. Reconstitution assays demonstrated that Thr-450 is important for regulating IL-2R complex formation, recruitment of JAK3, and activation of AKT and ERK1/2 and a transcriptionally active STAT5. These results provide the first evidence of the identification and functional characterization for threonine phosphorylation of an interleukin receptor.

Sensitivity of imatinib-resistant T315I BCR-ABL CML to a synergistic combination of ponatinib and forskolin treatment.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the life expectancy of patients suffering from chronic myeloid leukemia (CML); however, patients will eventually develop resistance to TKI therapy or adverse side effects due to secondary off-target mechanisms associated with TKIs. CML patients exhibiting TKI resistance are at greater risk of developing an aggressive and drug-insensitive disease. Drug-resistant CML typically arises in response to spontaneous mutations within the drug binding sites of the targeted oncoproteins. To better understand the mechanism of drug resistance in TKI-resistant CML patients, the BCR-ABL transformed cell line KCL22 was grown with increasing concentrations of imatinib for a period of 6 weeks. Subsequently, a drug-resistant derivative of the parental KCL22 cell line harboring the T315I gatekeeper mutation was isolated and investigated for TKI drug sensitivity via multi-agent drug screens. A synergistic combination of ponatinib- and forskolin-reduced cell viability was identified in this clinically relevant imatinib-resistant CML cell line, which also proved efficacious in other CML cell lines. In summary, this study provides new insight into the biological underpinnings of BCR-ABL-driven CML and potential rationale for investigating novel treatment strategies for patients with T315I CML.

Forskolin-inducible cAMP pathway negatively regulates T-cell proliferation by uncoupling the interleukin-2 receptor complex.

Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rß and γc chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.

Signal transducer and activator of transcription 5b (Stat5b) serine 193 is a novel cytokine-induced phospho-regulatory site that is constitutively activated in primary hematopoietic malignancies.

Signal transducer and activator of transcription 5b (Stat5b) is a critical node in the signaling network downstream of external (cytokines or growth factors) or internal (oncogenic tyrosine kinases) stimuli. Maximum transcriptional activation of Stat5b requires both tyrosine and serine phosphorylation. Although the mechanisms governing tyrosine phosphorylation and activation of Stat5b have been extensively studied, the role of serine phosphorylation remains to be fully elucidated. Using mass spectrometry and phospho-specific antibodies, we identified Ser-193 as a novel site of cytokine-induced phosphorylation within human Stat5b. Stat5b Ser(P)-193 was detected in activated primary human peripheral blood mononuclear cells or lymphoid cell lines in response to several γ common (γc) cytokines, including interleukin (IL)-2, IL-7, IL-9, and IL-15. Kinetic and spatial analysis indicated that Stat5b Ser-193 phosphorylation was rapid and transient and occurred in the cytoplasmic compartment of the cell prior to Stat5b translocation to the nucleus. Moreover, inducible Stat5b Ser-193 phosphorylation was sensitive to inhibitors of mammalian target of rapamycin (mTOR), whereas inhibition of protein phosphatase 2A (PP2A) induced phosphorylation of Ser-193. Reconstitution assays in HEK293 cells in conjunction with site-directed mutagenesis, EMSA, and reporter assays indicated that Ser(P)-193 is required for maximal Stat5b transcriptional activity. Indeed, Stat5b Ser-193 was found constitutively phosphorylated in several lymphoid tumor cell lines as well as primary leukemia and lymphoma patient tumor cells. Taken together, IL-2 family cytokines tightly control Stat5b Ser-193 phosphorylation through a rapamycin-sensitive mechanism. Furthermore, constitutive Ser-193 phosphorylation is associated with Stat5b proto-oncogenic activity and therefore may serve as a novel therapeutic target for treating hematopoietic malignancies.

Rapid Shift from SARS-CoV-2 Delta to Omicron Sub-Variants within a Dynamic Southern U.S. Borderplex.

COVID-19, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), remains an ongoing global health challenge. This study analyzed 3641 SARS-CoV-2 positive samples from the El Paso, Texas, community and hospitalized patients over 48 weeks from Fall 2021 to Summer 2022. The binational community along the U.S. southern border was predominantly SARS-CoV-2 Delta variant (B.1.617.2) positive for a 5-week period from September 2021 to January 2022 and quickly transitioned to the Omicron variant (B.1.1.529), which was first detected at the end of December 2021. Omicron replaced Delta as the predominant detectable variant in the community and was associated with a sharp increase in COVID-19 positivity rate, related hospitalizations, and newly reported cases. In this study, Omicron BA.1, BA.4, and BA.5 variants were overwhelmingly associated with S-gene dropout by qRT-PCR analysis unlike the Delta and Omicron BA.2 variants. The study reveals that a dominant variant, like Delta, can be rapidly replaced by a more transmissible variant, like Omicron, within a dynamic metropolitan border city, necessitating enhanced monitoring, readiness, and response from public health officials and healthcare workers.

Identification of a Unique Cytotoxic Thieno[2,3-c]Pyrazole Derivative with Potent and Selective Anticancer Effects In Vitro.

In recent years, the thienopyrazole moiety has emerged as a pharmacologically active scaffold with antitumoral and kinase inhibitory activity. In this study, high-throughput screening of 2000 small molecules obtained from the ChemBridge DIVERset library revealed a unique thieno[2,3-c]pyrazole derivative (Tpz-1) with potent and selective cytotoxic effects on cancer cells. Compound Tpz-1 consistently induced cell death at low micromolar concentrations (0.19 µM to 2.99 µM) against a panel of 17 human cancer cell lines after 24 h, 48 h, or 72 h of exposure. Furthermore, an in vitro investigation of Tpz-1's mechanism of action revealed that Tpz-1 interfered with cell cycle progression, reduced phosphorylation of p38, CREB, Akt, and STAT3 kinases, induced hyperphosphorylation of Fgr, Hck, and ERK 1/2 kinases, and disrupted microtubules and mitotic spindle formation. These findings support the continued exploration of Tpz-1 and other thieno[2,3-c]pyrazole-based compounds as potential small-molecule anticancer agents.

Identification of a Potent Cytotoxic Pyrazole with Anti-Breast Cancer Activity That Alters Multiple Pathways.

In this study, we identified a novel pyrazole-based derivative (P3C) that displayed potent cytotoxicity against 27 human cancer cell lines derived from different tissue origins with 50% cytotoxic concentrations (CC50) in the low micromolar and nanomolar range, particularly in two triple-negative breast cancer (TNBC) cell lines (from 0.25 to 0.49 µM). In vitro assays revealed that P3C induces reactive oxygen species (ROS) accumulation leading to mitochondrial depolarization and caspase-3/7 and -8 activation, suggesting the participation of both the intrinsic and extrinsic apoptotic pathways. P3C caused microtubule disruption, phosphatidylserine externalization, PARP cleavage, DNA fragmentation, and cell cycle arrest on TNBC cells. In addition, P3C triggered dephosphorylation of CREB, p38, ERK, STAT3, and Fyn, and hyperphosphorylation of JNK and NF-kB in TNBC cells, indicating the inactivation of both p38MAPK/STAT3 and ERK1/2/CREB signaling pathways. In support of our in vitro assays, transcriptome analyses of two distinct TNBC cell lines (MDA-MB-231 and MDA-MB-468 cells) treated with P3C revealed 28 genes similarly affected by the treatment implicated in apoptosis, oxidative stress, protein kinase modulation, and microtubule stability.

Protein phosphatase 2A regulates interleukin-2 receptor complex formation and JAK3/STAT5 activation.

Reversible protein phosphorylation plays a key role in interleukin-2 (IL-2) receptor-mediated activation of Janus tyrosine kinase 3 (JAK3) and signal transducer and activator of transcription 5 (STAT5) in lymphocytes. Although the mechanisms governing IL-2-induced tyrosine phosphorylation and activation of JAK3/STAT5 have been extensively studied, the role of serine/threonine phosphorylation in controlling these effectors remains to be elucidated. Using phosphoamino acid analysis, JAK3 and STAT5 were determined to be serine and tyrosine-phosphorylated in response to IL-2 stimulation of the human natural killer-like cell line, YT. IL-2 stimulation also induced serine/threonine phosphorylation of IL-2Rbeta, but not IL-2Rgamma. To investigate the regulation of serine/threonine phosphorylation in IL-2 signaling, the roles of protein phosphatase 1 (PP1) and 2A (PP2A) were examined. Inhibition of phosphatase activity by calyculin A treatment of YT cells resulted in a significant induction of serine phosphorylation of JAK3 and STAT5, and serine/threonine phosphorylation of IL-2Rbeta. Moreover, inhibition of PP2A, but not PP1, diminished IL-2-induced tyrosine phosphorylation of IL-2Rbeta, JAK3, and STAT5, and abolished STAT5 DNA binding activity. Serine/threonine phosphorylation of IL-2Rbeta by a staurosporine-sensitive kinase also blocked its association with JAK3 and IL-2Rgamma in YT cells. Taken together, these data indicate that serine/threonine phosphorylation negatively regulates IL-2 signaling at multiple levels, including receptor complex formation and JAK3/STAT5 activation, and that this regulation is counteracted by PP2A. These findings also suggest that PP2A may serve as a therapeutic target for modulating JAK3/STAT5 activation in human disease.

Phosphorylation of CrkL S114 induced by common gamma chain cytokines and T-cell receptor signal transduction.

T-cell activation and cellular expansion by common gamma chain cytokines such as Interleukin-2 is necessary for adaptive immunity. However, when unregulated these same pathways promote pathologies ranging from autoimmune disorders to cancer. While the functional role of Interleukin-2 and downstream effector molecules is relatively clear, the repertoire of phosphoregulatory proteins downstream of this pathway is incomplete. To identify phosphoproteins downstream of common gamma chain receptor, YT cells were radiolabeled with [32P]-orthophosphate and stimulated with Interleukin-2. Subsequently, tyrosine phosphorylated proteins were immunopurified and subjected to tandem mass spectrometry-leading to the identification of CrkL. Phosphoamino acid analysis revealed concurrent serine phosphorylation of CrkL and was later identified as S114 by mass spectrometry analysis. S114 was inducible through stimulation with Interleukin-2 or T-cell receptor stimulation. Polyclonal antibodies were generated against CrkL phospho-S114, and used to show its inducibility by multiple stimuli. These findings confirm CrkL as an Interleukin-2 responsive protein that becomes phosphorylated at S114 by a kinase/s downstream of PI3K and MEK/ERK signaling.

The Many Faces of JAKs and STATs Within the COVID-19 Storm.

The positive-sense single stranded RNA virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), resulted in a global pandemic with horrendous health and economic consequences not seen in a century. At a finer scale, immunologically, many of these devastating effects by SARS-CoV-2 can be traced to a "cytokine storm" resulting in the simultaneous activation of Janus Kinases (JAKs) and Signal Transducers and Activators of Transcription (STAT) proteins downstream of the many cytokine receptor families triggered by elevated cytokines found in Coronavirus Disease 2019 (COVID-19). In this report, cytokines found in the storm are discussed in relation to the JAK-STAT pathway in response to SARS-CoV-2 and the lessons learned from RNA viruses and previous Coronaviruses (CoVs). Therapeutic strategies to counteract the SARS-CoV-2 mediated storm are discussed with an emphasis on cell signaling and JAK inhibition.

Pharmacodynamic biomarkers in metronomic chemotherapy: multiplex cytokine measurements in gastrointestinal cancer patients.

Metronomic chemotherapy has shown promising antitumor activity in a number of malignancies. We previously reported a phase II clinical trial of metronomic UFT (a 5-fluorouracil prodrug; 100 mg/twice per day p.o.) and cyclophosphamide (CTX; 500 mg/m2 i.v. bolus on day 1 and then 50 mg/day p.o.) plus celecoxib (200 mg/twice a day p.o.) in 38 patients with advanced refractory gastrointestinal tumors. The mechanisms of action of metronomic chemotherapy include inhibition of angiogenesis, direct cytotoxic effects on cancer cells, and, at least for drugs such as CTX, activation of the immune system. To further evaluate the latter, we carried out an immune system multiplex 14-cytokine profiling of plasma samples that were available (for day 0, day 28, and day 56) from 31 of the 38 patients in the above-noted clinical trial. Our results show that pre-treatment plasma-level cutoffs of interferon gamma (> 12.84 pg/ml), sCD40L (< 2168 pg/ml), interferon alpha 2 (> 55.11 pg/ml), and IL-17a (< 15.1 pg/ml) were predictive markers for those patients with better progression-free survival (p < .05 for each cytokine). After 28 days of metronomic therapy, the plasma levels of sCD40L, IL-17a, and IL-6 (< 130 pg/ml) could serve as predictors of improved progression-free survival, as could levels interferon gamma and sCD40L after 56 days of therapy. We observed minimal changes in cytokine profiles, from baseline, as a consequence of the metronomic therapy, with the exception of an elevation of IL-6 and IL-8 levels 28 days (and 56 days) after treatment started (p < 0.05). Our results indicate that a selective cytokine elevation involves IL-6 and IL-8, following metronomic chemotherapy administration. In addition, interferon gamma and sCD40L may be potential biomarkers for gastrointestinal cancer patients that are likely to benefit from metronomic chemotherapy. Our study contributes to our understanding of the mechanisms of action of metronomic chemotherapy, and the cytokine profiling we describe may guide future selection of gastrointestinal cancer patients for UFT/CTX/celecoxib combination metronomic chemotherapy.

STAT5 regulation of BCL10 parallels constitutive NFkappaB activation in lymphoid tumor cells.

BACKGROUND: Signal Transducer and Activator of Transcription 5 A and B (STAT5) are key survival factors in cells of the lymphoid lineage. Identification of novel, tissue-specific STAT5 regulated genes would advance the ability to combat diseases due to aberrant STAT5 signaling. In the present work a library of human STAT5 bound genomic elements was created and validated. RESULTS: Of several STAT5 responsive genomic regulatory elements identified, one was located within the first intron of the human BCL10 gene. Chromatin immuno-precipitation reactions confirmed constitutive in vivo STAT5 binding to this intronic fragment in various human lymphoid tumor cell lines. Interestingly, non-phosphorylated STAT5 was found in the nuclei of Kit225 and YT cells in the absence of cytokine stimulation that paralleled constitutive NFkappaB activation. Inhibition of the hyperactive JAK3/STAT5 pathway in MT-2 cells via the Mannich-base, NC1153, diminished the constitutive in vivo occupancy of BCL10-SBR by STAT5, reduced NFkappaB activity and BCL10 protein expression in a dose dependent manner. Moreover, depletion of STAT5 via selective antisense oligonucleotide treatment similarly resulted in decreased BCL10 mRNA and protein expression, cellular viability and impaired NFkappaB activity independent of IL-2. CONCLUSION: These results suggest that the NFkappaB regulator BCL10 is an IL-2-independent STAT5 target gene. These findings proffer a model in which un-activated STAT5 can regulate pathways critical for lymphoid cell survival and inhibitors that disrupt STAT5 function independent of tyrosine phosphorylation may be therapeutically effective in treating certain leukemias/lymphomas.