Mukhopadhyay, Debabrata

Neuropilin-1 (NRP1), initially identified as a neuronal guidance protein, has emerged as a multifaceted regulator in cancer biology. Beyond its role in axonal guidance and angiogenesis, NRP1 is increasingly recognized for its significant impact on tumor progression and therapeutic outcomes. This review explores the diverse functions of NRP1 in cancer, encompassing its influence on tumor cell proliferation, migration, invasion, and metastasis. NRP1 interacts with several key signaling pathways, including vascular endothelial growth factor (VEGF), semaphorins, and transforming growth factor-beta (TGF-β), modulating the tumor microenvironment and promoting angiogenesis. Moreover, NRP1 expression correlates with poor prognosis in various malignancies, underscoring its potential as a prognostic biomarker. Therapeutically, targeting NRP1 holds promise as a novel strategy to inhibit tumor growth and enhance the efficacy of regular treatments such as chemotherapy and radiotherapy. Strategies involving NRP1-targeted therapies, including monoclonal antibodies, small molecule inhibitors, and gene silencing techniques, are being actively investigated in preclinical and clinical settings. Despite challenges in specificity and delivery, advances in understanding NRP1 biology offer new avenues for personalized cancer therapy. Although several types of cancer cells can express NRPs, the role of NRPs in tumor pathogenesis is largely unknown. Future investigations are needed to enhance our understanding of the effects and mechanisms of NRPs on the proliferation, apoptosis, and migration of neuronal, endothelial, and cancer cells. The novel frameworks or multi-omics approaches integrate data from multiple databases to better understand cancer’s molecular and clinical features, develop personalized therapies, and help identify biomarkers. This review highlights the pivotal role of NRP1 in cancer pathogenesis and discusses its implications for developing targeted therapeutic approaches to improve patient outcomes, highlighting the role of OMICS in targeting cancer patients for personalized therapy.

Heart failure with preserved ejection fraction (HFpEF), characterized by cardiac diastolic dysfunction, leads to premature mortality with limited effective treatments. A recent study in HFpEF patients suggested neuropilin 1 (NRP1), an endothelial cell (EC) originated prognostic marker, might contribute to the pathogenesis of HFpEF. However, the functional role of NRP1 in the pathogenesis of HFpEF remains unclear. Purpose: To determine the functional role of NRP1 in the pathogenesis of HFpEF. Hypothesis: NRP-1 contributes to the pathogenesis of HFpEF, potentially mediated through chronic inflammation. Methods: HFpEF was induced by hypertensive and metabolic stresses in control (n=14) and tamoxifen-induced, endothelial cell-specific NRP1 knockout (NRP1 iEC-KO ) mice (n=13). We assessed cardiac diastolic function via echocardiography and pressure-volume loop through invasive catheters. To determine the inflammation level, we tested leukocyte adhesion, inflammatory marker vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) mRNA level in control and NRP1 shRNA knockdown (NRP1 KD) human umbilical vein endothelial cells (HUVECs) with a stimulation of TNF-α. Student t-test (two tailed) was used and the criterion level of significance was set at p<0.05. Results: We found a significantly improved diastolic function in NRP1 iEC-KO mice, evidenced by a significantly reduced end diastolic pressure in NRP1 iEC-KO mice (8±2mmHg; n=7) compared to the controls (17±2mmHg; n=9; p=0.01) after HFpEF induction. E/A ratio was also significantly lower in NRP1 iEC-KO mice (2.0±0.2; n=10) compared to controls (2.7±0.3; n=12; p=0.04). Moreover, the isovolumic relaxation time was also significantly reduced in NRP1 iEC-KO mice (18±0.8ms; n=13) compared to controls (20±0.8ms; n=14; p=0.03). In addition, In HUVECs, NRP1 KD significantly reduced inflammatory marker VCAM-1 mRNA expression (95±16; n=5) compared to controls (293±36; n=5; p<0.01) upon TNF-α treatment. Similarly, NRP1 KD significantly reduced ICAM-1 mRNA expression (44±14; n=5) compared to controls (83±9; n=5; p=0.03) upon TNF-α treatment. Furthermore, leukocyte adhesion was significantly lower in the NRP1 KD group (57.0 ±8.6 cells/field) compared to the control group (90.8 ±9.1 cells/field; p=0.04). Conclusion: Endothelial NRP1 contributes to cardiac diastolic dysfunction and the pathogenesis of HFpEF, potentially mediated through the activation of chronic low-grade inflammation.

Recent advancements in disease modeling for myocardial infarction (MI) and coronary artery disease (CAD) have significantly enhanced our understanding of cardiovascular pathology and therapeutic development. This review comprehensively reviews the integration of in vivo and in vitro approaches to better model and study these conditions. We specifically focus on cutting-edge in vitro techniques, such as cardiac organoids, engineered heart tissues, and patient-derived induced pluripotent stem cells (iPSCs), which allow for detailed exploration of cellular and molecular mechanisms involved in MI and CAD. These models provide insights into ischemic injury, myocardial remodeling, and the effects of potential therapeutic interventions at a cellular level. In parallel, we discuss advances in the in vivo models, including genetically modified mice and large animal models, which offer valuable information on disease progression, cardiac function, and response to treatments within a more complex physiological context. By bridging these in vivo and in vitro approaches, researchers can gain a more comprehensive understanding of disease mechanisms, validate experimental findings, and accelerate the development of effective therapies. This review highlights recent progress, identifies current limitations, and proposes strategies for future research to enhance the translation of model-based discoveries into clinical practice for MI and CAD.

Melanoma is the most aggressive subtype of cancer, with a higher propensity to spread compared to most solid tumors. The application of OMICS approaches has revolutionized the field of melanoma research by providing comprehensive insights into the molecular alterations and biological processes underlying melanoma development and progression. This review aims to offer an overview of melanoma biology, covering its transition from primary to malignant melanoma, as well as the key genes and pathways involved in the initiation and progression of this disease. Utilizing online databases, we extensively explored the general expression profile of genes, identified the most frequently altered genes and gene mutations, and examined genetic alterations responsible for drug resistance. Additionally, we studied the mechanisms responsible for immune checkpoint inhibitor resistance in melanoma.

Abstract Gold nanoparticles for therapeutic interventions have become a widely popular source due to the ease in surface chemistry available for modification. Several studies undertaken focusses on the size, shape of nanoparticles as well as the signaling mechanisms and yet, knowledge regarding the alteration in membrane dynamics such as nanomechanical properties and cytokines involved during their uptake has not been studies before. To overcome the knowledge deficit, we employed atomic force microscopy and qRT-PCR techniques to acquire nanomechanical attributes and cytokines expressions respectively, in Panc-1 and AsPC-1 cell lines treated with surface modified gold nanoparticles for 1- and 24-hours’ time point. In nanomechanics paradigm, impetus was given on both linear attributes such as membrane stiffness, deformation, and adhesion as well as non-linear attributes such as drained Poisson’s ratio, diffusion coefficient and pore size. On the other hand, qRT-PCR technique yielded alterations in relative mRNA expressions of pro-tumorigenic cytokines such as CCL2, CXCL1, CXCL2, CXCL3, IL8, IL11, IL18 and TGFβ-1. Further, using Pearson’s correlation, we deduced the dependency between nanomechanical properties and cytokines expressions. We evaluated several criteria such as receptor dependent vs independent, PEGylated vs non-PEGylated and 1 hr vs 24hrs, to deduce correlations between cytokines and nanomechanical attributes. In Panc-1 cell line, we observed that PEGylation vs non-PEGylation of gold nanoparticles demonstrated most significant correlations, whereas in AsPC-1 cell line, receptor independent mechanism played a pivotal role in establishing significant correlations. For instance, D_C and P_D were highly correlated with CXCL1, CXCL2 and TGFβ-1 when non-PEGylated gold nanoparticles were up taken by Panc-1 cells. Similarly, D_C and P_D were highly correlated consistently with our cytokine panel except for TGFβ-1. In AsPC-1 cell line, deformation was significantly correlated with CCL2, CXCL2, CXCL3 and IL-8 whereas, D_C and P_D were significantly correlated with CCL2, CXCL1, CXCL3, IL-18 and TGFβ-1 in receptor independent scenario. This study demonstrated that during the uptake of gold nanoparticles, non-linear nanomechanical attributes are detrimental over linear attributes. Moreover, Surface modified gold nanoparticles induce varying levels of alteration in pro-tumorigenic cytokines. Taken together, this study boosts our insights into correlations describing cytokines regulating nanomechanical attributes in endocytosis paradigm. Citation Format: Tanmay Kulkarni, Enfeng Wang, Ramcharan Singh Angom, Debabrata Mukhopadhyay, Santanu Bhattacharya. Insights into correlation between nanomechanical properties and cytokines expression during gold nanoparticle endocytosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 496.

Abstract Background Renal cell carcinoma (RCC) was historically considered to be less responsive to radiation therapy (RT) compared to other cancer indications. However, advancements in precision high-dose radiation delivery through single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) have led to better outcomes and reduced treatment-related toxicities, sparking renewed interest in using RT to treat RCC. Moreover, numerous studies have revealed that certain therapeutic agents including chemotherapies can increase the sensitivity of tumors to RT, leading to a growing interest in combining these treatments. Here, we developed a rational combination of two radiosensitizers in a tumor-targeted liposomal formulation for augmenting RT in RCC. The objective of this study is to assess the efficacy of a tumor-targeted liposomal formulation combining the mTOR inhibitor everolimus (E) with the survivin inhibitor YM155 (Y) in enhancing the sensitivity of RCC tumors to radiation. Experimental Design: We slightly modified our previously published tumor-targeted liposomal formulation to develop a rational combination of E and Y in a single liposomal formulation (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further investigated how well EY-L sensitizes RCC cell lines and tumors toward radiation and explored the underlying mechanism of radiosensitization. Results EY-L outperformed the corresponding single drug-loaded formulations E-L and Y-L in terms of containing primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. EY-L also exhibited significantly higher sensitization of RCC cells towards radiation in vitro than E-L and Y-L. Additionally, EY-L sensitized RCC tumors towards radiation therapy in xenograft and murine RCC models. EY-L mediated induction of mitotic catastrophe via downregulation of multiple cell cycle checkpoints and DNA damage repair pathways could be responsible for the augmentation of radiation therapy. Conclusion Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy via inhibition of DNA damage repair and a substantial increase in mitotic catastrophe. This combination therapy may find its use in the augmentation of radiation therapy during the treatment of RCC patients.

Introduction: Genome-wide association studies (GWAS) have discovered >300 associations for CAD. Few loci are functionally characterized and may represent new mechanisms of disease. Hypothesis: Can an unbiased transcriptional screen for the >2000 candidate GWAS genes in endothelial cells (ECs) identify causal genes and disease-relevant pathways at CAD GWAS loci. Methods: We applied CRISPRi-Perturb-seq to knock down expression of genes within 500 Kb of coronary artery disease GWAS loci (2,300 genes in total) and measure their transcriptomic effects using single-cell RNA-seq. Validation of candidate causal genes was conducted in primary human ECs and zebrafish. Results: We identified 50 programs of co-expressed genes, which represent core cellular pathways (i.e. ribosome biogenesis) and 13 EC-specific pathways such as flow response and angiogenesis. The EC-specific programs show the greatest contribution to CAD heritability. 5 EC-specific programs have the greatest number of GWAS candidate genes. We find that 43 of 306 CAD GWAS signals converge onto these 5 gene programs, and all are regulated by genes in the cerebral cavernous malformations (CCM) pathway—which coordinates KLF2/4 transcriptional responses in endothelial cells, but has not been linked to CAD risk. The strongest regulator of these programs is TLNRD1 , which we show is a new CAD gene and novel regulator of the CCM pathway. TLNRD1 loss-of-function alters actin organization and barrier function in endothelial cells in vitro , and heart development in zebrafish in vivo . Conclusions: High-throughput functional analysis of 2,300 genes proximal to CAD GWAS loci prioritized pathways—such as angiogenesis and EC migration—that are regulated by multiple risk SNPs. Our study identifies new genes that likely influence risk for CAD, identifies convergence of CAD genes into certain pathways in endothelial cells, and prioritizes study of genes in the CCM-signaling pathway for contribution to CAD risk.

BACKGROUND: CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology-mediated genome editing has significantly improved the targeted inactivation of genes in vitro and in vivo in many organisms. Neuropilins play crucial roles in zebrafish heart regeneration, heart failure in mice, and electrical remodeling after myocardial infarction in rats. But the cell-specific functions of nrp1 have not been described before. In this study, we have investigated the role of nrp1 isoforms, including nrp1a and nrp1b , in cardiomyocytes during cardiac injury and regeneration in adult zebrafish hearts. METHODS: In this study, we have reported a novel CRISPR-based vector system for conditional tissue-specific gene ablation in zebrafish. Specifically, the cardiac-specific cmlc2 promoter drives Cas9 expression to silence the nrp1 gene in cardiomyocytes in a heat-shock inducible manner. This vector system establishes a unique tool to regulate the gene knockout in both the developmental and adult stages and hence widens the possibility of loss-of-function studies in zebrafish at different stages of development and adulthood. Using this approach, we investigated the role of neuropilin isoforms nrp1a and nrp1b in response to cardiac injury and regeneration in adult zebrafish hearts. RESULTS: We observed that both the isoforms ( nrp1a and nrp1b ) are upregulated after the cryoinjury. Interestingly, the nrp1b knockout significantly delayed heart regeneration and impaired cardiac function in the adult zebrafish after cryoinjury, demonstrated by reduced heart rate, ejection fractions, and fractional shortening. In addition, we show that the knockdown of nrp1b but not nrp1a induces activation of the cardiac remodeling genes in response to cryoinjury. CONCLUSIONS: To our knowledge, this study is novel where we have reported a heat-shock–mediated conditional knockdown of nrp1a and nrp1b isoforms using CRISPR/Cas9 technology in the cardiomyocyte in zebrafish and furthermore have identified a crucial role for the nrp1b isoform in zebrafish cardiac remodeling and eventually heart function in response to injury.

Abstract Purpose: Although renal cell carcinoma (RCC) is relatively more resistant to radiation therapy (RT) compared to other cancer indications, recent advances in single-fraction and multi-fraction stereotactic ablative radiotherapy (SABR) made it possible to achieve better outcome and reduce treatment related toxicities. In addition, with studies demonstrating that certain therapeutic agents including chemotherapies can act as radiosensitizers, the idea of combining radiotherapy with those agents are being actively pursued. Among them, mammalian target of rapamycin (mTOR) inhibitor everolimus (E) and survivin inhibitor YM155 (Y) are of special interest since they target multiple RT-resistance mechanisms simultaneously. Additionally, YM155 has been shown to help in overcoming resistance to mTOR inhibitors in various cancer including RCC. Hence, the present study aims to evaluate the efficacy of a tumor-targeted liposomal formulation combining everolimus and YM155 (EY-L) in sensitizing RCC tumors towards radiation. Experimental Design: Previously we developed a tumor-targeted liposomal formulation and used it successfully to deliver multiple drugs for the treatment of RCC in both immune-deficient and immune-competent mouse models. Here we developed a similar formulation combining everolimus and YM155 (EY-L) and assessed its efficacy in RCC cell lines in vitro and in RCC tumors in vivo. We further evaluated the efficacy of EY-L in sensitizing RCC cell lines and tumors towards radiation in vitro using clonogenic assay and in vivo in RCC xenografts in immunodeficient mice and murine RCC models in immunocompetent mice. Results: EY-L (2 mg/kg E, 1.4 mg/kg Y, twice a week intravenous administration) was better than E-L or Y-L, the corresponding single drug loaded formulations in containing the primary tumor growth and improving survival in an immunocompetent syngeneic mouse model of RCC. Furthermore, EY-L leads to significantly higher sensitization of RCC cells towards radiation in vitro than E-L or Y-L. Finally, EY-L was able to sensitize RCC tumors towards radiation therapy in both RCC xenograft and murine RCC model. Conclusion: Taken together, our study demonstrated the efficacy of a strategic combination therapy in sensitizing RCC to radiation therapy using in vitro clonogenic assay and in vivo radiosensitization studies in multiple RCC tumor models. Citation Format: Krishnendu Pal, Vijay S. Madamsetty, Hari K. Rachamala, Shamit K. Dutta, Enfeng Wang, Debabrata Mukhopadhyay. Combined inhibition of mammalian target of rapamycin (mTOR) and survivin augments radiation therapy in renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B026.