Biology subjects

Sriram, G.

Publications and source records attributed to Sriram, G..

6 recordsLinked to original sources

bioRxiv2026

Live immunogenic melanoma cells generated by etoposide treatment upregulate B2M and lose tumor-forming potential

Cytotoxic chemotherapy has been the primary cancer treatment modality for decades, however, chemotherapy drugs do not selectively target cancer cells. These drugs are toxic to all proliferating cells including immune cells that constitute the host-defense system against pathogens and cancer cells. In an effort to develop non-toxic combinations of chemotherapy and immunotherapies for cancer, we previously reported in Sriram et al. 2021 that etoposide-treated B16 melanoma cells expressing ovalbumin, when co-cultured in vitro with dendritic cells and T-cells, enhance T-cell activation. The live fraction of melanoma cells after etoposide treatment was critical to promote T-cell activation as dead cells alone or in combination with cell-free supernatants were not effective. In this study, we show that the live fraction of etoposide-treated B16-Ova cells, which gain the ability to stimulate T-cells, are growth-arrested, but remain viable past the co-incubation period with dendritic cells and T-cells post etoposide washout. Furthermore, using a soft-agar colony-formation assay as a proxy for tumor-forming potential, we show that these live etoposide-treated B16-Ova cells have drastically reduced ability to form colonies displaying a near-complete loss of anchorage-independent growth. We also show that etoposide treatment upregulates the production of Beta-2-microglobulin (B2M), a subunit of Class I MHC. Intriguingly, we observed using immunofluorescence that the live etoposide-treated B16-Ova cells show enhanced intracellular perinuclear B2M accumulation but B2M surface staining, while detectable, is not enhanced by etoposide treatment. Furthermore, while the live etoposide-treated B16-Ova cells express Class I MHC protein, there is only a marginal drug treatment-induced increase observed in the expression levels by western blotting. Our results suggest that T-cell activation by live etoposide-treated B16-Ova cells is likely not driven by drug treatment-induced enhancement in cell surface levels of Class I MHC and point to other mechanisms.

cancer biology↗

bioRxiv2025

Structural Identification of Major Molecular Determinants for Phosphotyrosine Recognition in Tyrosine Kinases Reveals Tumour Promoting and Suppressive Functions

Protein tyrosine kinases activate signaling pathways by catalyzing the phosphorylation of tyrosine residues in their substrates. Mounting evidence suggests that, in addition to recognizing phosphorylated tyrosine (pTyr) residues through specific phosphobinding modules, many protein kinases selectively recognize pTyr directly adjacent to the tyrosine residue they phosphorylate and catalyze the formation of twin pTyr-pTyr sites. Here, we demonstrate the importance of this phosphopriming-driven twin pTyr signaling in promoting cell cycle progression through the cell cycle-inhibitory protein p27Kip1. We identify, structurally resolve, and tune two distinct molecular determinants driving the selective recognition of pTyr directly N- and C-terminal to the target phospho-acceptor tyrosine site. We further show structural and biochemical conservation in this recognition, and identify cancer-associated alterations to these determinants that are unable to recognize phosphoprimed substrates. Finally, using an in vivo mouse model of leukemia we show that Bcr-Abl mutants unable to recognize phosphoprimed substrates paradoxically result in enhanced tumor development and progression. These data indicate that Bcr-Abl, like other proto-oncogenes such as Ras or Myc, engages both pro- and anti-oncogenic programs - but in the case of Bcr-Abl, this is accomplished through a mechanism involving traditional and phosphoprimed substrate recognition.

biochemistry↗

bioRxiv2023

Cell-free chromatin particles activate immune checkpoints in human T cells: Implications for cancer therapy

Immune checkpoint blockade is an exciting breakthrough in cancer therapy, but how immune checkpoints are activated is unknown. We have earlier reported that cell-free chromatin particles (cfChPs) that circulate in the blood, or those that are released locally from dying cells, are readily internalized by healthy cells with biological consequences. Here we show that treatment of human lymphocytes with cfChPs isolated from sera of cancer patients led to marked activation of immune checkpoints viz. PD-1, CTLA-4, LAG-3, NKG2A, and TIM-3. Concurrently activated were stress-related markers cJun, cFos, JunB, FosB, NF[Kcy]B, and EGR1. The above immune checkpoints were also activated when lymphocytes were treated with cfChPs released from dying HeLa cells; the latter could be abrogated by three cfChPs deactivating agents. These results suggest that immune checkpoints are activated by lymphocytes as stress response to cfChPs. Simultaneous downregulation of multiple immune checkpoints may herald a new approach to immunotherapy of cancer. Statement of SignificanceWe show that cell-free chromatin particles (cfChPs) that circulate in the blood of cancer patients, or those released from dying cancer cells, simultaneously activate five immune checkpoints as a stress response by human lymphocytes. Activation of checkpoints was abrogated by cfChPs deactivating agents suggesting a novel approach to cancer treatment.

immunology↗

bioRxiv2023

Therapeutic interventions on human xenografts promote systemic dissemination of oncogenes

We generated xenografts of human cancer cells in mice, and using immuno-FISH analysis detected multiple co-localizing signals of human DNA and eight human oncoproteins in brain cells. Signals increased dramatically five days after treatment with chemotherapy, localized radiotherapy or surgery, which could be minimized by concurrent treatment with cell-free chromatin deactivating agents. These results suggest that therapeutic interventions may potentially encourage metastatic spread which is preventable by deactivating cell-free chromatin.

cancer biology↗

bioRxiv2020

Bacterially produced small molecules stimulate diatom growth

Diatoms are photosynthetic microalgae that fix a significant fraction of the worlds carbon. Because of their photosynthetic efficiency and high-lipid content, diatoms are priority candidates for biofuel production. Here, we report that sporulating Bacillus thuringiensis when in co-culture with a marine diatom Phaeodactylum tricornutum significantly increases the diatom cell count. Bioassay-guided purification led to the identification of two diketopiperazines (DKPs) that both stimulate P. tricornutum growth and increase its lipid content. RNA-seq analysis revealed upregulation of a small set of P. tricornutum genes involved in iron starvation response and nutrient recycling when DKP was added to the diatom culture. This work demonstrates that two DKPs produced by a bacterium could positively impact P. tricornutum growth and lipid content, offering new approaches to enhance P. tricornutum-based biofuel production. As increasing numbers of DKPs are isolated from marine microbes, the work gives potential clues to bacterially produced growth factors for marine microalgae. One sentence summaryTwo diketopiperazines (DKPs) produced by sporulating bacterium Bacillus thuringiensis stimulate diatom P. tricornutum growth and increase diatom lipid content.

plant biology↗

bioRxiv2020

The Injury Response to DNA Damage Promotes Anti-Tumor Immunity

Inhibition of immune checkpoints has shown promising results in the treatment of certain tumor types. However, the majority of cancers do not respond to immune checkpoint inhibition (ICI) treatment, indicating the need to identify additional modalities that enhance the response to immune checkpoint blockade. In this study, we identified a tumor-tailored approach using ex-vivo DNA damaging chemotherapy-treated tumor cells as a live injured cell adjuvant. Using an optimized ex vivo system for dendritic cell-mediated T-cell IFN-{gamma} induction in response to DNA-damaged tumor cells, we identified specific dose-dependent treatments with etoposide and mitoxantrone that markedly enhance IFN-{gamma} production by T-cells. Unexpectedly, the immune-enhancing effects of DNA damage failed to correlate with known markers of immunogenic cell death or with the extent of apoptosis or necroptosis. Furthermore, dead tumor cells alone were not sufficient to promote DC cross-presentation and induce IFN-{gamma} in T-cells. Instead, the enhanced immunogenicity resided in the fraction of injured cells that remained alive, and required signaling through the RIPK1, NF-kB and p38MAPK pathways. Direct in vivo translation of these findings was accomplished by intra-tumoral injection of ex vivo etoposide-treated tumor cells as an injured cell adjuvant, in combination with systemic anti-PD1/CTLA4 antibodies. This resulted in increased intra-tumoral CD103+ dendritic cells and circulating tumor antigen-specific CD8+ T-cells, leading to enhanced anti-tumor immune responses and improved survival. The effect was abrogated in BATF3-deficient mice indicating that BATF3+ DCs are required for appropriate T-cell stimulation by live but injured DNA-damaged tumor cells. Notably, injection of the free DNA-damaging drug directly into the tumor failed to elicit such an enhanced anti-tumor response as a consequence of simultaneous damage to dendritic cells and T-cells. Finally, the DNA damage induced injured cell adjuvant and systemic ICI combination, but not ICI alone, induced complete tumor regression in a subset of mice who were then able to reject tumor re-challenge, indicating induction of a long-lasting anti-tumor immunological memory by the injured cell adjuvant treatment in vivo.

cancer biology↗