Rational Target Research Articles

RNAseq correlative biomarkers IFIT1B and VSTM5 predict progression free survival and clinical benefit in a multi-site Phase I/II trial of Olaparib and Tremelimumab for gBRCAm recurrent ovarian cancer (LBA 11)

<b>Objectives:</b> Although ovarian cancer is a rational target for immune therapy, immune checkpoint blockade has not demonstrated the efficacy seen in other solid tumors. In preclinical studies, PARP inhibition (PARPi) synergized with CTLA4 immune checkpoint blockade, resulting in durable anti-tumor immunity and long-term survival in ovarian cancer models. This combination was tested in women with germline BRCA1 or BRCA2 mutations (gBRCAm) and recurrent ovarian cancer in a phase I/II clinical trial (NCT0257125). Correlative studies were performed to identify predictive biomarkers of response. <b>Methods:</b> Women with recurrent ovarian cancer, confirmed gBRCAm, and measurable disease were eligible for participation. There were no restrictions on the number or type of prior treatment regimens. Prior PARPi treatment was allowed. Subjects received olaparib at 300mg BID and the CTLA4 antibody tremelimumab at 10mg/kg IV every 4 weeks for six doses followed by maintenance dosing every 12 weeks. Treatment continued for up to two years. The Phase I lead-in allowed dose modifications for toxicity. The phase II component used Simon's 2-stage design based on the primary endpoint of overall response rate. RNAseq was performed on archived formalin-fixed, paraffin embedded pre-treatment tumor samples from 30 subjects, and candidate biomarkers were selected based on differential expression in subjects with and without clinical benefit. <b>Results:</b> Forty-nine patients received study treatment (mean age 56 yrs, range 39-79) at five sites. Twenty-eight subjects had received ≥3 prior chemotherapy regimens, and 29 were previously treated with a PARPi. After 14 patients received the study regimen, the recommended phase 2 schedule for tremelimumab was changed to 10mg/kg infusions every 4 weeks for four doses followed by maintenance dosing every 12 weks due to immune-related adverse events, primarily colitis. Fifty-nine percent of subjects experienced at least one non-hematologic ≥ grade 3 toxicity. Among 44 evaluable patients, the overall response rate was 39%, with a clinical benefit rate of 48%. The median PFS was 3.4 months (95% CI 2.7-6.1). In a curated analysis of interferon response genes, lack of IFIT1B expression was associated with significantly enhanced PFS (median 11.4 vs 2.7 months; log-rank p=0.009; Fig 1). In an unbiased analysis of all genes, VSTM5 was the most differentially expressed marker (AUC=0.852), and lack of VSTM5 expression was significantly associated with PFS (8.12 vs 2.56 months; log-rank p=0.001). <b>Conclusions:</b> The combination of olaparib and tremelimumab achieved durable treatment benefit in a subset of gBRCAm recurrent ovarian cancer patients. Immune toxicity was moderate. Biomarkers based on gene expression in archived tumor samples were associated with clinical benefit. Future studies are needed to assess the utility of these biomarkers to identify candidates for immune therapy.

P418: MODELLING AND TARGETING ACUTE MYELOID LEUKAEMIA CELLS IN THE BONE MARROW PROTECTIVE NICHE

Background: AML is a therapeutic challenge due to its aggressiveness and biological heterogeneity. Although 80% of patients initially achieve a complete remission, the long-term disease-free survival is poor. One issue contributing to disease relapse, is persistence of disease in the protective niche of the Bone Marrow microenvironment (BMME). Here, AML cells are surrounded by other cell types that promote their survival, which enables them to evade therapeutic destruction and promotes the emergence of drug resistance. Clinical trials of single agent mobilising drugs, like Plerixafor, have yielded promising results but they are not curative. So, the development of combinatorial therapies that simultaneously target different components of AML cell adhesion may release more AML cells into the circulation, where they can be targeted by standard therapies. Aims: a) To develop a multi-cellular, co-culture system to recapitulate the adhesive BMME, b) To test rational drug combinations and identify targets with the potential to mobilise anchored AML cells and c) To perform paired transcriptomic and phenotypic analysis of persistently adhered versus mobilised AML cells to detect novel targets. Methods: We have developed a novel 96-well plate multi-cellular, co-culture system using stromal cells (HS5), endothelial cells (HUVEC), osteoblasts (hFOB 1.19) and AML cells (OCI-AML3 and KG1a). Multiple mobilisation drugs, alone and in combination, were tested and AML cell release was quantified by flow cytometry. Drugs tested to date include: Plerixafor (CXCR4 inhibitor), Natalizumab (anti-α4-integrin antibody), ONO-7161 (novel CXCR4 inhibitor), purified anti-CD44 and anti-E-Selectin. Phenotypic and transcriptional comparisons were made between the persistently adherent and mobilised AML cells. Results: The most adhesive physiological “BMME mix” was identified as HS5, HUVEC and hFOB 1.19 in equal 1/3 proportions (p=0.0001). Co-culture with AML cells resulted in 74% and 68% adherence of KG1a and OCI-AML3, respectively. Phenotypic analysis of adhered vs non-adhered AML cells identified CD44, CXCR4, CD49d and CD38 as important markers of optimal BMME adhesion. Subsequently, our novel co-culture system was used as a drug testing platform to assess clinically available agents. Plerixafor and Natalizumab increased detachment by 1.3-fold (p=0.0132) and 1.2-fold (p=0.015) respectively. In contrast, ONO7161 and anti-E-selectin had little effect. The most promising candidate, anti-CD44, resulted in a 2-fold and 1.6-fold detachment of KG1a (p=0.004) and OCI-AML3 (p=0.027), respectively. However, even in the presence of the maximum dose of anti-CD44 (5µg/mL)), 46% of KG1a and 48% of OCI-AML3 cells remained persistently adhered. Synergy experiments with anti-CD44 in combination with either Plerixafor or Natalizumab yielded no better release than anti-CD44 alone. Summary/Conclusion: We have developed a novel, multi-cellular co-culture system that recapitulates the adhesive BMME. We are using this as a drug testing platform for AML to identify the most potent release agents and test the effects of cytotoxic drugs on those most persistently adhered. To-date, anti-CD44 is the most effective release agent in our model, but it still failed to release all AML cells. Transcriptomic analysis of the persistently adherent AML cells should enable us to delineate the critical biological interactions required for BMME adhesion. This will enable us to identify rational new targets to effectively remove chemo-resistant AML cells from the protective BM niche.

Redefinition of Successful Treatment of Patients With Hypothyroidism. Is TSH the Best Biomarker of Euthyroidism?

In recent years evidence has accumulated supporting a revised view of the nature of euthyroidism and the biomarkers of thyroid function. Within the normal range, variations in thyroid hormone levels are associated with variations in clinical parameters and outcomes. There are therefore no readily identified individually specific optimum levels of thyroid hormones for any individual. Levels around the middle of the normal population range may best reflect euthyroidism. These levels may have evolutionary advantages on the basis that adverse outcomes often increase with divergence from such levels, and physiological processes tend to minimise such inter-individual and intra-individual divergence. In populations of predominantly untreated individuals, levels of thyroid hormones and in particular levels of free thyroxine (FT4) correlate more often with clinical parameters than do levels of thyrotropin (TSH). Levels of thyroid hormones may therefore be regarded as the best available biomarkers of euthyroidism and dysthyroidism. It follows that ‘subclinical hypothyroidism’ (normal FT4/raised TSH levels), rather than being an accurate marker of peripheral tissue hypothyroidism is more a marker of decreased thyroid reserve and prognosis. The recent evidence suggests that treatment of hypothyroxinemia, regardless of the TSH level, and monitoring therapy using FT4 and/or triiodothyronine levels, depending on the replacement regime, may result in more successful treatment of hypothyroidism than relying on thyrotropin levels for patient selection and subsequent treatment monitoring. The equivalents of mid-range levels of thyroid hormones (especially FT4), adjusted by individual comorbidity concerns, may be rational general replacement targets. These implications of the new evidence may create opportunities for novel trials of thyroid replacement therapy.

Cathepsin B Gene Knockout Improves Behavioral Deficits and Reduces Pathology in Models of Neurologic Disorders.

Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.

The effect of novel synthetic semicarbazone- and thiosemicarbazone-linked 1,2,3-triazoles on the apoptotic markers, VEGFR-2, and cell cycle of myeloid leukemia

Vascular Endothelial Growth Factor II (VEGFR-2) has been proved as a rational target in cancer therapy. Although currently prescribed VEGFR-2 inhibitors are showing potent antitumor activity, they are often causing serious unwanted effects, restricting their extensive use as chemotherapeutics. Herein, after analyzing the structures of the effective VEGFR-2 inhibitor molecules, we report the synthesis of a new set of semicarbazone- and thiosemicarbazone-linked 1,2,3-triazoles with expected potency of inhibiting the VEGFR-2 signaling. The design of new compounds considered maintaining the essential pharmacophoric features of sorafenib for effective binding with the receptor target. All compounds have been evaluated for their growth inhibition effect against a panel of sixty cancer cells at the National Cancer Institute. Leukemia cancer cells, especially HL-60 and SR, were shown to be the most sensitive to the cytotoxic effect of new compounds. Thiosemicarbazones 21, 26, and 30 exhibited the best activity against almost all tested cancer cells. Therefore, a set of subsequent in vitro biological evaluations has been performed to understand the mechanistic effect of these compounds further. They inhibited the VEGFR-2 with IC50 values of 0.128, 0.413, and 0.067µM respectively compared with 0.048µM of Sorafenib. The probable mechanistic effect of 30 has been further evaluated on a number of apoptotic and antiapoptotic markers including BAX, BCL2, caspase-3, and caspase-9. Results revealed the potential of the thiosemicarbazone-linked triazole 30 to induce both the early and the late apoptosis, elevate BAX/BCL2 ratio, induce caspase-3 & caspase-9, and arrest the HL-60 cell cycle at the G2/M and G0-G1 phases. Molecular docking of new semicarbazones and thiosemicarbazones into the proposed biological target receptor has also been performed. Results of docking studies proved the potential of new semicarbazone- and thiosemicarbazone-linked 1,2,3-triazoles to effectively bind with crucial residues of the VEGFR-2 binding pocket.

Abstract 5491: Targeting importin-YAP axis in pancreatic ductal adenocarcinoma

Abstract Background: To date, pancreatic ductal adenocarcinoma (PDAC) remains to have a dismal prognosis, with a 5-year survival rate of only 10%, which brings out an imperative to develop new therapeutic strategies to improve patient outcome. Recently, aberrant nucleocytoplasmic transport machinery in cancer cells has emerged as a rational therapeutic target. We aim to validate the nuclear importin complex involved in the import of macromolecules across the nuclear membrane as a potential therapeutic target in PDAC. Methods: Tet-inducible shRNA and FDA-approved agent (Ivermectin) were used for the inhibition of importins. Cell death (apoptosis and pyroptosis) was detected by western blot. In vivo tumor growth of human and mouse PDAC cells was evaluated in immunocompetent and immunodeficient animal models. MTT assay was employed to evaluate the patient-derived organoid viability following drug treatment. Results: We found that PDAC cells/patients harbored unusually higher levels of importins. Inducible knockdown of importin-α2/α5 (KPNA2/KPNA1) or importin-β1 (KPNB1) strongly suppressed PDAC tumor growth and induced apoptosis and pyroptosis. Interestingly, the importin inhibitor Ivermectin (an FDA-approved antiparasitic agent) worked effectively in PDAC cells, patient-derived organoids and animal models. Mechanistically, we show that importins associate with YAP (the transcriptional coactivator of the Hippo pathway) and control its expression at mRNA levels. In line with these observations, the expression levels of importin-αs, importin-β1, and YAP are positively correlated in PDAC patients. Conclusion: Our study identifies the importin complex as a prognostic marker and therapeutic target for pancreatic cancer and is expected to provide preclinical evidence for the evaluation of Ivermectin in PDAC patients. Citation Format: Renya Zeng, Jixin Dong. Targeting importin-YAP axis in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5491.

Abstract LB213: Potent antitumor activity of a FGFR4 CAR-T in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. Despite multi-modal aggressive therapies, relapsed, refractory or metastatic rhabdomyosarcoma remains a lethal disease with no significant improvement in outcome over decades of clinical trials. Therefore novel therapies are needed. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase that is overexpressed in RMS when compared with normal tissues, and mutationally activated in about 7.5% of RMS. Recently we showed that PAX3-FOXO1 establishes a super-enhancer in the FGFR4 genomic locus driving its high expression in fusion positive RMS. CAR T-cell therapy is effective in treating refractory and relapsed B-cell leukemia and lymphoma, with three CARs targeting CD19 approved by the FDA. Multiple CART trials are currently underway for solid tumors. Since FGFR4 is a cell surface protein, we hypothesized that FGFR4 will provide a rational target for immunotherapy in RMS. We confirmed by immunohistochemistry staining, western analysis, and Meso Scale Discovery that FGFR4 protein is highly differentially expressed in RMS samples. We developed a murine anti-FGFR4 antibody, 3A11, by immunizing mouse with FGFR4-IG fusion protein. 3A11 showed high affinity and specificity of binding to FGFR4. We then developed a second-generation CAR using the VL and VH domain of 3A11 antibody and found that the scFvFc retained its specificity and high affinity at nanomolar range. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out or FGFR4 negative cells. 3A11 CART incubated with human primary cells obtained from liver, kidney, heart, and pancreas, did not elicit a cytokine response, indicating a low potential for “on-target off-tumor” toxicity. In vivo testing also found that 3A11 CART eliminated RMS cells in both murine xenograft metastatic and localized subcutaneous models. Therefore we have developed a CART targeting FGFR4 that shows high potency for treating RMS. A phase 1 FGFR4-CART clinical trial is planned for children and adolescent young adults with relapsed/refractory rhabdomyosarcoma. Citation Format: Adam Tai Chi Cheuk, Meijie Tian, Nityashree Shivaprasad, Steven Highfill, David Milewski, G Tom Brown, Peter Azorsa, Dina Schneider, Berkley Gryder, Jun S Wei, Young Kwok Song, Hsien-Chao Chou, Jerry Wu, Joon-Yong Chung, Brian Belyea, Corinne Linardic, Stephen M Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent antitumor activity of a FGFR4 CAR-T in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB213.

TBIO-08. The molecular basis for rational targeting of FGFR-driven growth and invasiveness in pediatric brain tumors

Abstract The oncogenic activation of receptor tyrosine kinases (RTK) promotes growth, survival and dissemination in pediatric tumors including glioma, ependymoma and medulloblastoma (MB). Direct targeting of either the RTK or of downstream kinases can effectively block tumor promoting pathway functions. However, emergence of resistance is common. We hypothesized that alternative interference strategies that target protein-protein interactions (PPIs) instead of enzymatic activities could overcome the emergence of resistance. We characterized the molecular interactions downstream of the FGFR that regulate relevant growth and invasion-promoting mechanisms in MB cells, to identify potentially druggable PPIs. We found that the FRS2 protein is an essential up-stream effector of FGFR signaling towards invasiveness. Using a proteomics approach, we furthermore identified the Striatin 3 protein as a novel oncogenic effector of the FGFR pathway downstream of FRS2, as it integrates antagonistic growth and invasion signals downstream of FGFR. Mechanistically, Striatin 3 interacts with the Ser/Thr kinase MAP4K4, couples it to the protein phosphatase 2A, and thereby inactivates growth repressing activities of MAP4K4. In parallel, Striatin 3 enables MAP4K4-mediated phosphorylation of PKC-theta and VASP, which combined are necessary to promote tissue invasion. To selectively repress pro-invasive FGFR functions, we identified and functionally validated small molecule ligands of FRS2, that prevent FRS2 activation and downstream signaling. We demonstrate efficacy of these compounds in inhibiting invasion and growth promoting activities in vitro and in vivo, and identified potential off-target activities of the ligand using a proteome-wide interaction analysis. We propose inhibition of FRS2 by a small molecular PTB domain ligand as a strategy to repress FGF signaling in FGFR-driven tumors. The development of this ligand, and the de novo design of functional analogs thereof bear promise for further pre-clinical evaluation of these structures as anti-growth promoting and anti-metastatic therapeutics applicable to FGFR-driven tumors.

A Bioinformatic Rational Gene Targeting Approach to Treating Pathologies With Healthy Diet

ObjectivesWe hypothesize that identification of healthy whole foods that modulate disease-causing gene expression back toward the normal is a low-cost, healthy, and readily-translatable alternative and/or complementary approach to costly and sometimes toxic pharmaceutical drugs. Our objectives are (1) to identify human studies assessing gene expression after healthy whole food ingestion, and (2) use this data to develop an online app for patients, community and healthcare providers as a dietary guide for a wide range of diseases and conditions.MethodsWe used the keywords “human”, “gene expression” and separately, 42 different dietary agents with reported health benefits to search GEO, PubMed, Google Scholar, Clinical trials, Cochrane library, and EMBL-EBI for studies. For app development, The R-Shiny platform was utilized to construct a public web app (“Eat4Genes”) featuring: a drop-down disease or condition selector; risk genes; user menus; dietary studies; ranking; and study links. To identify key risk genes, the above databases, Big Pharma targets, and NCBI's “Genes and Disease” were reviewed.Results42 human whole food (or extract) studies were identified. Despite varying conditions (e.g., 4 hours to 8 week diets), the number of whole genome modulated genes were reasonable consistent (1061 +/− 227 SEM). Human gene expression studies were only found for 13 of 34 searched whole foods, underscoring the need for more as compared with much less useful but much more prevalent dietary supplement, cell culture and rodent studies. We identified 96 key risk genes for a wide range of pathologies (e.g., HMGCR for Hypercholesterolemia) for our app. Successful app construction was achieved with the above features, and included the ability to select specific diseases/conditions followed by their key risk genes, identification of dietary agents that express and modulate these genes, and the studies supporting these findings with numerous links. A usability survey was also carried out (N = 17).ConclusionsOur dietary rational gene targeting approach offers an innovative, low-cost, healthy, personalized, and readily translatable strategy to improve health and reduce treatment costs. Construction of our new Eat4Genes app now extends its promise to practice in the form of an interactive online dietary guide.Funding SourcesNIH, the Wildermuth Foundation, and IDEA at RPI.

P-81 Phase 3 study of MK4280A (coformulated favezelimab and pembrolizumab) versus standard of care in previously treated PD-L1–positive metastatic colorectal cancer (mCRC)

Immune checkpoint (IC) inhibitors are effective in mismatch repair-deficient or microsatellite instability-high colorectal cancer (CRC). However, for mismatch repair-proficient (pMMR) mCRC, which account for 95% of mCRC tumors, treatment options are limited. In patients with CRC, LAG-3 is overexpressed on colorectal immune cells and correlated with poor differentiation, advanced stage, lymph node involvement and invasion depth (Chen J, Zihua C. Med Oncol. 2014; 31:82). As such, LAG-3 represents a rational target for IC inhibition in CRC. IC inhibitor combination therapy with MK4280A, a coformulation of anti–LAG3 antibody favezelimab and pembrolizumab, has shown promising antitumor activity and a manageable safety profile in patients with PD-L1 CPS≥1 microsatellite stable mCRC and may be efficacious in pMMR mCRC (Garralda E et al. J Clin Oncol. 2021;39. Abstract 3584). In this ongoing phase 3 study (NCT05064059), we will evaluate the efficacy and safety of MK-4280A in patients with PD-L1–positive pMMR mCRC. The open-label, parallel group, active-controlled study plans to enroll approximately 432 patients aged ≥18 years with histologically confirmed unresectable mCRC, pMMR and PD-L1 CPS ≥1 tumors, and measurable disease per RECIST v1.1 by investigator review. All patients must have experienced progressive disease after: 1) fluoropyrimidine, irinotecan and oxaliplatin, with or without anti-VEGF antibody, anti–EGFR antibody for patients with left-sided tumors that are RAS WT, and 2) RAF inhibitor with or without binimetinib in patients with BRAF v600E mutations. Key eligibility criteria include ECOG status 0 or 1, adequate organ function, and availability of archival or newly obtained tissue sample to assess PD-L1 and MMR status. Patients will be randomly assigned to MK-4280A (coformulated favezelimab 800 mg/pembrolizumab 200 mg; Arm A) IV Q3W or standard of care (Arm B) (regorafenib 160 mg PO Q4W [QD on days 1-21; no dose on days 22-28] or TAS-102 35mg/m2 PO Q4W [BID on days 1-5 and 8-12; no dose on days 6, 7, and 13-28]). Patients will be stratified by geographic region (Asia Pacific, EMEA/Americas), presence of liver metastases (yes, no) and time from initial diagnosis of mCRC to randomization (≥18 mo, < 18 mo). Treatment will continue for ≤35 cycles of MK-4280A or unacceptable toxicity, disease progression, confirmed CR with MK-4280A must have been treated for ≥8 cycles and have received 2 cycles after the initial CR was observed (Arm A), or withdrawal from study. Disease assessment by CT or MRI will be performed Q9W during the study. Primary end point is overall survival (OS). Secondary end points are progression free survival (PFS), objective response rate (ORR) and duration of response (by blinded independent central review per RECIST v1.1), patient-reported outcomes on health-related quality of life, and safety graded per NCI CTCAE v5.0. OS and PFS will be estimated by Kaplan-Meier methods. ORR with 95% CI will be estimated using the Clopper-Pearson method. ClinicalTrials.gov, NCT05064059. Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA.

Effect of pH on stability of dimer structure of the main protease of coronavirus-2

The viral main protease (Mpro) from a novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a key enzyme essential for viral replication and has become an attractive target for antiviral drug development. The Mpro forms a functional dimer and exhibits a pH-dependent enzyme activity and dimerization. Here, we report a molecular dynamics (MD) investigation to gain insights into the structural stability of the enzyme dimer at neutral and acidic pH. Our data shows larger changes in structure of the protein with the acidic pH than that with the neutral pH. Structural analysis of MD trajectories reveals a substantial increase in intersubunit separation, the loss of domain contacts, binding free energy and interaction energy of the dimer which implies the protein instability and tendency of dimer dissociation at acidic pH. The loss in the interaction energy is mainly driven by electrostatic interactions. We have identified the intersubunit hydrogen-bonding residues involved in the decreased dimer stability. These findings may be helpful for rational drug design and target evaluation against COVID-19.

Inhibition of mitochondrial complex I reverses NOTCH1-driven metabolic reprogramming in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is commonly driven by activating mutations in NOTCH1 that facilitate glutamine oxidation. Here we identify oxidative phosphorylation (OxPhos) as a critical pathway for leukemia cell survival and demonstrate a direct relationship between NOTCH1, elevated OxPhos gene expression, and acquired chemoresistance in pre-leukemic and leukemic models. Disrupting OxPhos with IACS-010759, an inhibitor of mitochondrial complex I, causes potent growth inhibition through induction of metabolic shut-down and redox imbalance in NOTCH1-mutated and less so in NOTCH1-wt T-ALL cells. Mechanistically, inhibition of OxPhos induces a metabolic reprogramming into glutaminolysis. We show that pharmacological blockade of OxPhos combined with inducible knock-down of glutaminase, the key glutamine enzyme, confers synthetic lethality in mice harboring NOTCH1-mutated T-ALL. We leverage on this synthetic lethal interaction to demonstrate that IACS-010759 in combination with chemotherapy containing L-asparaginase, an enzyme that uncovers the glutamine dependency of leukemic cells, causes reduced glutaminolysis and profound tumor reduction in pre-clinical models of human T-ALL. In summary, this metabolic dependency of T-ALL on OxPhos provides a rational therapeutic target.

A Highly Sensitive and Specific Detection Method for Mycobacterium tuberculosis Fluoroquinolone Resistance Mutations Utilizing the CRISPR-Cas13a System.

ObjectivesCRISPR-Cas13a system-based nucleic acid detection methods are reported to have rapid and sensitive DNA detection. However, the screening strategy for crRNAs that enables CRISPR-Cas13a single-base resolution DNA detection of human pathogens remains unclear.MethodsA combined rational design and target mutation-anchoring CRISPR RNA (crRNA) screening strategy was proposed.ResultsA set of crRNAs was found to enable the CRISPR-Cas13 system to dramatically distinguish fluroquinolone resistance mutations in clinically isolated Mycobacterium tuberculosis strains from the highly homologous wild type, with a signal ratio ranging from 8.29 to 38.22 in different mutation sites. For the evaluation of clinical performance using genomic DNA from clinically isolated M. tuberculosis, the specificity and sensitivity were 100 and 91.4%, respectively, compared with culture-based phenotypic assays.ConclusionThese results demonstrated that the CRISPR-Cas13a system has potential for use in single nucleotide polymorphism (SNP) detection after tuning crRNAs. We believe this crRNA screening strategy will be used extensively for early drug resistance monitoring and guidance for clinical treatment.

Mechanism of tethered agonist-mediated signaling by polycystin-1

Polycystin-1 (PC1) is an important unusual G protein-coupled receptor (GPCR) with 11 transmembrane domains, and its mutations account for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 shares multiple characteristics with Adhesion GPCRs. These include a GPCR proteolysis site that autocatalytically divides these proteins into extracellular, N-terminal, and membrane-embedded, C-terminal fragments (CTF), and a tethered agonist (TA) within the N-terminal stalk of the CTF that is suggested to activate signaling. However, the mechanism by which a TA can activate PC1 is not known. Here, we have combined functional cellular signaling experiments of PC1 CTF expression constructs encoding wild type, stalkless, and three different ADPKD stalk variants with all-atom Gaussian accelerated molecular dynamics (GaMD) simulations to investigate TA-mediated signaling activation. Correlations of residue motions and free-energy profiles calculated from the GaMD simulations correlated with the differential signaling abilities of wild type and stalk variants of PC1 CTF. They suggested an allosteric mechanism involving residue interactions connecting the stalk, Tetragonal Opening for Polycystins (TOP) domain, and putative pore loop in TA-mediated activation of PC1 CTF. Key interacting residues such as N3074–S3585 and R3848–E4078 predicted from the GaMD simulations were validated by mutagenesis experiments. Together, these complementary analyses have provided insights into a TA-mediated activation mechanism of PC1 CTF signaling, which will be important for future rational drug design targeting PC1.

Prognostic stratification of HPV-associated oropharyngeal cancer based on CD103+ immune cell abundance in patients treated on TROG 12.01 and De-ESCALaTE randomized trials

High CD103+ intratumoral immune cell (ITIC) abundance is associated with better prognosis in unselected patients with human papilloma virus-associated oropharyngeal squamous cell carcinoma (HPV-associated OPSCC) treated with cisplatin and radiotherapy (CIS/RT). Substituting cetuximab (CETUX) for CIS with RT in HPV-associated OPSCC resulted in inferior efficacy. Our aim was to determine whether quantification of CD103 ITIC could be used to identify a population of HPV-associated OPSCC with superior prognosis. We pooled data from the TROG 12.01 and De-ESCALaTE randomized trials that compared CETUX/70GyRT with CIS/70GyRT in low-risk HPV-associated OPSCC: American Joint Committee on Cancer 7 stage III (excluding T1-2N1) or stage IV (excluding N2b-c if smoking history >10 pack-years and/or distant metastases), including all patients with available tumor samples. The primary endpoint was failure-free survival (FFS) in patients receiving CETUX/RT comparing CD103+ ITIC high (≥30%) versus low (<30%). High and low CD103 were compared using Cox regression adjusting for age, stage and trial. Tumor samples were available in 159/182 patients on TROG 12.01 and 145/334 on De-ESCALaTE. CD103+ ITIC abundance was high in 27% of patients. The median follow-up was 3.2 years. The 3-year FFS in patients treated with CETUX/RT was 93% [95% confidence interval (CI) 79% to 98%] in high CD103 and 74% (95% CI 63% to 81%) in low CD103 [adjusted hazard ratio= 0.22 (95% CI 0.12-0.41), P < 0.001]. The 3-year overall survival in patients treated with CETUX/RT was 100% in high CD103 and 86% (95% CI 76% to 92%) in low CD103, P < 0.001. In patients treated with CIS/RT, there was no significant difference in FFS. CD103+ ITIC expression separates CETUX/RT-treated low-risk HPV-associated OPSCC into excellent and poor prognosis subgroups. The high CD103 population is a rational target for de-intensification trials.

Fused in Liposarcoma Protein, a New Player in the Regulation of HIV-1 Transcription, Binds to Known and Newly Identified LTR G-Quadruplexes.

HIV-1 integrated long terminal repeat (LTR) promoter activity is modulated by folding of its G-rich region into non-canonical nucleic acids structures, such as G-quadruplexes (G4s), and their interaction with cellular proteins. Here, by a combined pull-down/mass spectrometry/Western-blot approach, we identified the fused in liposarcoma (FUS) protein and found it to preferentially bind and stabilize the least stable and bulged LTR G4, especially in the cell environment. The outcome of this interaction is the down-regulation of viral transcription, as assessed in a reporter assay with LTR G4 mutants in FUS-silencing conditions. These data indicate that the complexity and dynamics of HIV-1 LTR G4s are much greater than previously envisaged. The G-rich LTR region, with its diverse G4 landscape and multiple cell protein interactions, stands out as prime sensing center for the fine regulation of viral transcription. This region thus represents a rational antiviral target for inhibiting both the actively transcribing and latent viruses.

Acat‐1 Inhibition Reduces Inflammatory Response to Bleomycin‐mediated Lung Injury

Acute lung injury (ALI) is characterized by epithelial damage, barrier dysfunction, and pulmonary edema. Macrophage (MΦ) activation and failure to resolve play a role in in ALI, thus MΦ phenotype modulation is a rational target for therapeutic intervention. Large, lipid‐laden MΦs have been observed in various injury models, including intratracheal bleomycin (ITB), suggesting that lipid storage may play a role in ALI severity. The mitochondrial enzyme Acat‐1 is highly expressed in MΦs where it catalyzes the esterification of cholesterol, leading to intracellular lipid accumulation. We hypothesize that inhibition of Acat‐1 will reduce MΦ activation and reduce ITB‐mediated lung injury. K‐604, a selective inhibitor of Acat‐1, was used to reduce lipid accumulation in ITB. Male and female C57BL6/J mice (n=16‐21/group) were administered PBS, K‐604, ITB, or ITB +K‐604 on d0, PBS or K‐604 on d3, and were sacrificed on d7. ITB caused significant (*p&lt;0.05 v PBS) body weight loss (‐13.92±2.05%* v 2.66±0.78%) and an increase in cholesterol accumulation (71.63±11.7 μM* v 15.51±9.1 μM) and size (29.67±10.6 μm* v 11±4.3 μm) of pulmonary MΦs. These changes were mitigated by Acat‐1 inhibition, as ITB+K‐604 mice had significantly (#p&lt;0.05 v ITB) reduced body weight loss (4.04±1.03%#), cholesterol accumulation (13.79±5.4#), and MΦ diameter (14.0±3.7 μm#), confirming successful inhibition of Acat‐1 in the lung. Histological analysis showed that K‐604 significantly reduced ITB‐induced alveolar wall thickening (2.3±0.05 μm# v 4.35±0.04 μm) and reduced measures of consolidation and cell infiltration. Surface active function was normalized as indicated by a significant decrease in phospholipid:SP‐B ratio in ITB+K‐604 compared to ITB (7.95±1.22# v 13.28±2.17). K‐604 administration rescued ITB‐mediated loss of resident alveolar MΦs (CD45+/Siglec F+/F/480+, CD11c+, CD11b‐) (72.00±5.67%# v 49.58±8.52%) and was found to decrease the % of pro‐inflammatory alveolar MΦs (CD45+/Siglec F+/F/480+, Ly6c+, CD206‐) (3.86±0.88%# v 8.22±1.76%). K‐604 reduced ITB‐mediated increases in NOS2 (64.23±20.07# v 131.89±17.07) and ARG1 expression (42.39±8.14# v 177.41±52.2) in BAL MΦs, suggesting reduced inflammatory signaling and immunometabolic demand. In our ITB model, K‐604 also decreased the % of interstitial MΦs (IMs) (tissue‐derived CD45+, Siglec F ‐, F4/80+, CD11b+) expressing markers of maturity and fibrotic potential (CD11c+/CD206+) (5.89±2.15%# v 19.72±1.93%). IMs were also found to have an increased rate of lipid uptake after K‐604 regardless of ITB administration as measured by the uptake of fluorescently labeled LDL and visualization by confocal microscopy. These data, along with changes in cholesterol ester accumulation, suggest that Acat‐1 inhibition alters lipid storage and uptake within pulmonary macrophages, affecting cell phenotype and function. Thus, Acat‐1 may be a target for intervention in ALI.

Broadly neutralizing antibodies against Omicron-included SARS-CoV-2 variants induced by vaccination

The SARS-CoV-2 Omicron variant shows substantial resistance to neutralization by infection- and vaccination-induced antibodies, highlighting the demands for research on the continuing discovery of broadly neutralizing antibodies (bnAbs). Here, we developed a panel of bnAbs against Omicron and other variants of concern (VOCs) elicited by vaccination of adenovirus-vectored COVID-19 vaccine (Ad5-nCoV). We also investigated the human longitudinal antibody responses following vaccination and demonstrated how the bnAbs evolved over time. A monoclonal antibody (mAb), named ZWD12, exhibited potent and broad neutralization against SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa, Delta, and Omicron by blocking the spike protein binding to the angiotensin-converting enzyme 2 (ACE2) and provided complete protection in the challenged prophylactic and therapeutic K18-hACE2 transgenic mouse model. We defined the ZWD12 epitope by determining its structure in complex with the spike (S) protein via cryo-electron microscopy. This study affords the potential to develop broadly therapeutic mAb drugs and suggests that the RBD epitope bound by ZWD12 is a rational target for the design of a broad spectrum of vaccines.

Polyamines in cancer: integrating organismal metabolism and antitumour immunity.

The natural mammalian polyamines putrescine, spermidine and spermine are essential for both normal and neoplastic cell function and replication. Dysregulation of metabolism of polyamines and their requirements is common in many cancers. Both clinical and experimental depletion of polyamines have demonstrated their metabolism to be a rational target for therapy; however, the mechanisms through which polyamines can establish a tumour-permissive microenvironment are only now emerging. Recent data indicate that polyamines can play a major role in regulating the antitumour immune response, thus likely contributing to the existence of immunologically 'cold' tumours that do not respond to immune checkpoint blockade. Additionally, the interplay between the microbiota and associated tissues creates a tumour microenvironment in which polyamine metabolism, content and function can all be dramatically altered on the basis of microbiota composition, dietary polyamine availability and tissue response to its surrounding microenvironment. The goal of this Perspective is to introduce the reader to the many ways in which polyamines, polyamine metabolism, the microbiota and the diet interconnect to establish a tumour microenvironment that facilitates the initiation and progression of cancer. It also details ways in which polyamine metabolism and function can be successfully targeted for therapeutic benefit, including specifically enhancing the antitumour immune response.

Reactogenicity, safety, and immunogenicity of chimeric haemagglutinin influenza split-virion vaccines, adjuvanted with AS01 or AS03 or non-adjuvanted: a phase 1–2 randomised controlled trial

One strategy to develop a universal influenza virus vaccine is to redirect the immune system to the highly conserved haemagglutinin stalk domain by sequentially administering vaccines expressing chimeric (c) haemagglutinins with a conserved stalk domain and divergent head domain, to which humans are naive. We aimed to assess the reactogenicity, safety, and immunogenicity of adjuvanted and unadjuvanted investigational supra-seasonal universal influenza virus vaccines (SUIVs) in healthy young adults. In this observer-masked, randomised, controlled, phase 1-2 trial, we recruited adults aged 18-39 years with no clinically significant conditions from six centres in Belgium and the USA. Participants were randomly assigned to ten equally sized groups via an online system with the MATerial Excellence programme. Vaccines contained heterosubtypic group 1 H8, H5, or H11 haemagglutinin heads, an H1 haemagglutinin stalk, and an N1 neuraminidase (cH8/1N1, cH5/1N1, and cH11/1N1; haemagglutinin dose 15 μg/0·5 mL), administered on days 1 and 57, with a month 14 booster. SUIVs were evaluated in the sequences: cH8/1N1-placebo-cH5/1N1, cH5/1N1-placebo-cH8/1N1, or cH8/1N1-cH5/1N1-cH11/1N1, adjuvanted with either AS03 or AS01, or not adjuvanted. The last group received inactivated quadrivalent influenza vaccine (IIV4)-placebo-IIV4. Primary outcomes were safety (analysed in the exposed population) and immunogenicity in terms of the anti-H1 stalk humoral response at 28 days after vaccination (analysed in the per-protocol population, defined as participants who received the study vaccines according to the protocol). This trial is registered with ClinicalTrials.gov, NCT03275389. Between Sept 25, 2017, and March 26, 2020, 507 eligible participants were enrolled. 468 (92%) participants received at least one dose of study vaccine (exposed population), of whom 244 (52%) were included in the per-protocol population at final analysis at month 26. The safety profiles of all chimeric vaccines were clinically acceptable, with no safety concerns identified. Injection-site pain was the most common adverse event, occurring in 84-96% of participants receiving an adjuvanted SUIV or non-adjuvanted IIV4 and in 40-50% of participants receiving a non-adjuvanted SUIV. Spontaneously reported adverse events up to 28 days after vaccination occurred in 36-60% of participants, with no trends observed for any group. 17 participants had a serious adverse event, none of which were considered to be causally related to the vaccine. Anti-H1 stalk antibody titres were highest in AS03-adjuvanted groups, followed by AS01-adjuvanted and non-adjuvanted groups, and were higher after cH8/1N1 than after cH5/1N1 and after a two-dose primary schedule than after a one-dose schedule. Geometric mean concentrations by ELISA ranged from 21 938·1 ELISA units/mL (95% CI 18 037·8-26 681·8) in the IIV4-placebo-IIV4 group to 116 596·8 ELISA units/mL (93 869·6-144 826·6) in the AS03-adjuvanted cH8/1N1-cH5/1N1-cH11/1N1 group 28 days after the first dose and from 15 105·9 ELISA units/mL (12 007·7-19 003·6) in the non-adjuvanted cH5/1N1-placebo-cH8/1N1 group to 74 639·7 ELISA units/mL (59 986·3-92 872·6) in the AS03-adjuvanted cH8/1N1-cH5/1N1-cH11/1N1 group 28 days after the second dose. The stalk domain seems to be a rational target for development of a universal influenza virus vaccine via administration of chimeric haemagglutinins with head domains to which humans are naive. GlaxoSmithKline Biologicals.