Tanmoy Mondal

Receptor clustering is the most critical step to activate extrinsic apoptosis by death receptors belonging to the TNF superfamily. Although clinically unsuccessful, using agonist antibodies, the death receptors-5 remains extensively studied from a cancer therapeutics perspective. However, despite its regulatory role and elevated function in ovarian and other solid tumors, another tumor-enriched death receptor called Fas (CD95) remained undervalued in cancer immunotherapy until recently, when its role in off-target tumor killing by CAR-T therapies was imperative. By comprehensively analyzing structure studies in the context of the binding epitope of FasL and various preclinical Fas agonist antibodies, we characterize a highly significant patch of positively charged residue epitope (PPCR) in its cysteine-rich domain 2 of Fas. PPCR engagement is indispensable for superior Fas agonist signaling and CAR-T bystander function in ovarian tumor models. A single-point mutation in FasL or Fas that interferes with the PPCR engagement inhibited apoptotic signaling in tumor cells and T cells. Furthermore, considering that clinical and immunological features of the autoimmune lymphoproliferative syndrome (ALPS) are directly attributed to homozygous mutations in FasL, we reveal differential mechanistic details of FasL/Fas clustering at the PPCR interface compared to described ALPS mutations. As Fas-mediated bystander killing remains vital to the success of CAR-T therapies in tumors, our findings highlight the therapeutic analytical design for potentially effective Fas-targeting strategies using death agonism to improve cancer immunotherapy in ovarian and other solid tumors.

TPS6106 Background: Around 85% of head and neck cancer patients in India are seen in locally advanced stage which either relapse or fail after the first line treatment. The options of systemic therapy treatment in second line (after first line palliative chemotherapy or failure within 6 months of chemotherapy as part of multimodality treatment) are limited. The median progression free survival (PFS) of 1-3 months and median overall survival (OS) of 4-6 months are reported across various studies. There is need for having effective medical treatment option. Rationale- Triple metronomic is oral, cheap and requires minimum resources. We will be comparing physician choice of standard systemic therapies with ASCO recommended triple metronomic therapy. Methods: Design- Open label randomized, superiority. Study Period- 4 years. Inclusion- Head and Neck cancer patients (Age > 18 years) either platinum refractory or planned for second line (ECOG PS ≤2 and adequate organ function). Exclusion- Uncontrolled comorbidities, Pregnancy & lactating women. Baseline - blood parameters, EORTC QOL & HN module and baseline axial imaging. Central stratified randomization (Computer generated randomization sheet) will be done- ; the stratification will be for site of tumor (oral cavity versus others) and ECOG PS (0-1/2). Arm A: Triple metronomic (Per Oral )- Erlotinib 150 mg once daily, Celecoxib 200 mg twice daily & Weekly Methotrexate 9 mg/m². Arm B: Physician choice therapy- Docetaxel, Paclitaxel, Cetuximab, Nivolumab, Afatinib, Pembrolizumab, 5-Flurouracil, Capecitabine Therapy will be continued till progression or intolerable side effects. Adverse events monitoring as per the NCI CTCAE 5 criteria. The response will be monitored in accordance with institutional standards. Quality of life (EORTC) would be collected at baseline, at 2 months and at 6 months. The 6 month OS assumed on the basis of previous data of docetaxel was 55, we assume that it would increase with a target hazard ratio 0.561 with type 1 error of 5%, type 2 error of 20% and 10% lost to follow up rate. The sample size is 114. Events required for analysis-93. Outcome measures & Statistical analysis:OS- from date of randomization to date of death. Patients alive at their last follow ups would be censored. PFS from date of randomization to date of progression or death whichever is earlier. Patients who have not progressed at their last follow ups would be censored. OS and PFS will be estimated by Kaplan meier analysis and would be compared between the arms by the log rank test. Cox proportional hazard model would be constructed for calculation of hazard ratio. The model would also be used to see the impact of chemotherapy regimen on overall survival in accordance with known prognostic factors. Age (below or above 60 years), gender, site, hemoglobin level (equal to or below 12 g/dl or above it) and PS (0-1 versus 2). To compare the QOL scores between the 2 arms. Clinical trial information: CTRI/2021/08/036002.

Since its emergence in December 2019, COVID-19 has remained a global economic and health threat. Although RNA and DNA vector-based vaccines induced antibody response and immunological memory have proven highly effective against hospitalization and mortality, their long-term efficacy remains unknown against continuously evolving SARS-CoV-2 variants.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor‐enriched death receptor‐5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase‐II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD‐L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase‐8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD‐L1 stability on tumor cell surface. Targeting DR5‐ROCK1‐PD‐L1 axis markedly increases immune effector T‐cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism.

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1–conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention.Significance: TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Cancer is a major health problem and the most upsetting disease in humans, leading to death in both developed and developing countries. Proper treatment of the disease is still a challenge. Chemotherapy is considered one of the regimens to cure this disease. In this study, a series of 1,2,3-triazole and isoxazole-linked benzothiazole/benzoxazole derivatives were synthesized and evaluated for their anticancer activity against human cancer cell lines, such as HeLa (cervical), and A549 (lung) cell lines, with HEK-293 cell line used as a control. Among them, conjugates 8a, 8f, 13g, 13h and 13j displayed significant cytotoxic activity against human cancer cell lines. Furthermore, these active conjugates induced an increase in the expression of key apoptotic genes that are involved in the intrinsic pathway of apoptosis such as caspase-9, caspase-3, BAX and cytochrome-c. This study may provide possible anti-cancer therapeutics against HeLa and lung cancer cells.

Argonaute family proteins are well conserved among all organisms. Its role in mitotic cell cycle progression and apoptotic cell elimination is poorly understood. Earlier we have established the contribution of Ago-1 in cell cycle control related to G2/M cyclin in Drosophila. Here we have extended our study in understanding the relationship of Ago-1 in regulating apoptosis during Drosophila development. Apoptosis play a critical role in controlling organ shape and size during development of multi cellular organism. Multifarious regulatory pathways control apoptosis during development among which highly conserved JNK (c-Jun N-terminal kinase) pathway play a crucial role. Here we have over expressed Ago-1 in Drosophila eye and brain by employing UAS (upstream activation sequence)-GAL4 system under the expression of eye and brain specific driver. Over expression of Ago-1 resulted in reduced number of ommatidia in the eye and produced smaller size brain in adult and larval Drosophila. A drastic reversal of the phenotype towards normal was observed upon introduction of a single copy of the dominant negative mutation of basket (bsk, Drosophila homolog of JNK) indicating an active and physical involvement of the bsk with Ago-1 in inducing developmental apoptotic process. Further study showed that Ago-1 stimulates phosphorylation of JNK through transforming growth factor-β activated kinase 1- hemipterous (Tak1-hep) axis of JNK pathway. JNK phosphorylation results in up regulation of pro-apoptotic genes head involution defective (hid), grim & reaper (rpr) and induces activation of Drosophila caspases (cysteinyl aspartate proteinases);DRONC (Death regulator Nedd2-like caspase), ICE (alternatively Drice, Death related ICE-like caspase) and DCP1 (Death caspase-1) by inhibiting apoptotic inhibitor protein DIAP1 (Death-associated inhibitor of apoptosis 1). Further, Ago-1 also inhibits miR-14 expression to trigger apoptosis. Our findings propose that Ago-1 acts as a key regulator in controlling cell death, tumor regression and stress response in metazoan providing a constructive bridge between RNAi machinery and cell death.