Biology subjects

Peeper, D. S.

Publications and source records attributed to Peeper, D. S..

8 recordsLinked to original sources

bioRxiv2025

Decoding interaction-induced proteome changes in co-cultures with hybrid quantification and SILAC-directed real-time search

Intercellular communication between T cells and cancer cells plays a pivotal role in determining cancer cell survival or death. Yet, our understanding of this interaction remains incomplete. Methods to study heterotypic cell interactions are either limited to targeted studies relying on predefined set of proteins, or require cell separation, thus disrupting the native environment. Stable isotope labeling by amino acids in cell culture (SILAC) enables proteome distinction in heterologous co-cultures without the need for physical separation. But dynamic studies remain constrained by the need for numerous mass spectrometry (MS) runs, the challenges in detecting low-abundant proteins, particularly in immune cells and the limited data completeness due to the use of data-dependent MS1-based precursor quantification. To overcome these limitations, we evaluate the integration of SILAC with tandem mass tag (TMT) multiplexing and SILAC-directed real-time search (RTS). TMT labeling enables simultaneous analysis of multiple samples, while RTS-MS3 acquisition using SILAC-induced mass shifts as fixed modifications triggers MS3 scans for specific proteome populations within a mixed cell system, improving quantitative accuracy and proteome coverage for target protein populations. We benchmarked our acquisition methods using SILAC-labeled samples mixed at defined ratios and validated the approach in biologically relevant co-culture experiments. Additionally, we introduced a carrier channel to enhance detection of lower-abundant T cell proteins, while maintaining acceptable quantitative precision. Our results demonstrate that the combined SILAC-TMT-RTS strategy dramatically improves proteome depth, temporal resolution, and cell-type specificity for short-term co-culture interaction proteomics studies. In co-culture samples of T cells with non-small cell lung cancer cell lines that were either sensitive or resistant to T cell killing, our method revealed candidate mechanisms underlying their differential sensitivity. Our integrated approach combining SILAC, TMT and RTS to resolve cell-specific proteome dynamics in co-culture represents a novel and powerful advance.

cancer biology↗

bioRxiv2025

Inhibition of TBK1/IKKe mediated RIPK1 phosphorylation sensitizes tumors to immune cell killing

Resistance to immune cell-mediated cytotoxicity poses a significant challenge in cancer therapy, compromising the efficacy of immunotherapeutic approaches such as immune checkpoint blockade (ICB) treatment. To enhance therapy outcomes, it is crucial to identify interventions that can synergize with ICB therapy to overcome tumor resistance. Therefore, we need to define the cellular mechanisms that sensitize tumors to cytotoxic T cells. CD8 T cells rely on cytokines such as TNF to carry out their cytotoxicity against tumors, and recent findings link select tumor mutations in the TNF pathway to increased T cell killing, in a manner dependent on RIPK1 kinase. Here, we demonstrate that sensitized tumor cells fail to initiate inhibitory RIPK1 phosphorylation at site S25 upon T cell attack, thereby foregoing a pro-survival checkpoint early in TNF signal transduction. Consequently, tumor cells experiencing a loss of TNF-induced RIPK1 S25 phosphorylation exhibit increased RIPK1 activation and fail to recruit non-canonical IKK kinases (TBK1 and IKKe) to the TNFR1 complex. Functional knockouts of TBK1 and IKKe in melanoma cells result in heightened sensitivity not only in CD8 T cell but also in Natural Killer cell attacks. Our findings indicate that preventing TBK1 and IKKe recruitment to the TNF signaling complex, thereby blocking RIPK1 pro-survival phosphorylation and promoting direct RIPK1 activation, is a tractable strategy to increase tumor sensitivity to immune cell killing and has the potential to benefit current immunotherapy interventions.

cell biology↗

bioRxiv2025

Resistance to BRAF inhibitors drives melanoma sensitivity to Chk1 inhibition

BRAF inhibitor-resistant melanomas (BRAFiR) acquire (epi)genetic and functional alterations that enable them to evade alternative treatments. Identifying these alterations is critical to advancing treatment strategies. Here, we explored the effect of Chk1 inhibition (Chk1i) on BRAFiR cells, revealing higher sensitivity compared to treatment-naive cells both in vitro and in vivo. Using FUCCI-labeling and time-lapse microscopy, we show that S phase progression is required for Chk1i-induced cytotoxicity in BRAFiR cells, but not in treatment-naive cells. Replication stress markers, including reduced BrdU incorporation and increased phospho-RPA and {gamma}H2AX, were exclusive to BRAFiR cells exposed to Chk1i. Untreated BRAFiR cells exhibited upregulated DNA replication genes, reduced progressing forks and increased origin firing, suggesting intrinsic replication changes. MAPK pathway reactivation in treatment-naive cells mimicked BRAFiR traits, increasing sensitivity to Chk1i. These findings indicate that Chk1i exploits elevated replication stress specifically in BRAFiR, highlighting its therapeutic potential in overcoming MAPK inhibitor resistance in melanoma.

cancer biology↗

bioRxiv2023

Phosphoprotein dynamics of interacting tumor and T cells by HySic

Functional interactions between cytotoxic T cells and tumor cells are central to anti-cancer immunity. Some of the proteins involved, particularly immune checkpoints expressed by T cells, serve as promising clinical targets in immunotherapy. However, our understanding of the complexity and dynamics of the interactions between tumor cells and T cells is only rudimentary. Here we present HySic (for Hybrid quantification of SILAC (Stable Isotope Labelling by Amino acids in Cell culture)-labeled interacting cells) as an innovative method to quantify protein and phosphorylation dynamics between and within physically interacting (heterotypic) cells. We show that co-cultured HLA/antigen-matched tumor and T cells engage in physical and stable interactions, allowing for in-depth HySic analysis. This method does not require physical separation of the two cell types for subsequent MS proteome and phosphoproteome measurement using label free quantification (LFQ). We demonstrate that HySic can be used to unravel proteins contributing to functional T cell:tumor cell interactions. We validated HySic with established interactions, including those mediating IFN{gamma} signaling. Using HySic we identified the RHO/RAC/PAK1 signaling pathway to be activated upon interaction of T cells and tumor cells. Pharmacologic inhibition of PAK1 sensitized tumor cells to T cell killing. Thus, HySic is an innovative and simple method to study short-term protein signaling dynamics in physically interacting cells, which can be easily extended to other biological systems.

cancer biology↗

bioRxiv2022

TSC2 regulates tumor susceptibility to TRAIL-mediated T cell killing by orchestrating mTOR signaling

Resistance to cancer immunotherapy continues to impair common clinical benefit. Here, we uncover an important role for Tuberous Sclerosis Complex 2 (TSC2) in determining tumor susceptibility to cytotoxic T lymphocytes (CTL) killing, both in vitro and in vivo. TSC2-depleted tumor cells showed disrupted mTOR regulation upon CTL attack, which was associated with enhanced cell death. Tumor cells adapted to CTL attack by shifting their mTOR signaling balance toward increased mTORC2 activity to circumvent apoptosis and necroptosis. TSC2 critically protected tumor cells as its ablation strongly augmented tumor cell sensitivity to CTL attack. Mechanistically, TSC2 inactivation caused elevation of TRAIL receptors expression, cooperating with mTORC1-S6 signaling to induce tumor cell death. Clinically, we found a negative correlation between TSC2 expression and TRAIL signaling in TCGA patient cohorts. Moreover, a lower TSC2 immune response signature was observed in melanomas from patients responding to immune checkpoint blockades. Our study uncovers a pivotal role for TSC2 in the cancer immune response by governing crosstalk between TSC2-mTOR and TRAIL signaling, aiding future therapeutic exploration of this pathway in immuno-oncology.

cancer biology↗

bioRxiv2020

PREDICTIVE IMMUNE CHECKPOINT BLOCKADE CLASSIFIERS DISTINGUISHING MONO- VERSUS COMBINATION THERAPY REQUIREMENT

Although high clinical response rates are seen for immune checkpoint blockade (ICB) treatment of metastatic melanomas, both intrinsic and acquired ICB resistance remain considerable clinical challenges1. Combination ICB (anti-PD-1 + anti-CTLA-4) shows improved patient benefit2-5, but is associated with severe adverse events and exceedingly high cost. Therefore, there is a dire need to stratify individual patients for their likelihood of responding to either anti-PD-1 or anti-CTLA-4 monotherapy, or the combination. Since it is conceivable that ICB responses are influenced by both tumor cell-intrinsic and -extrinsic factors6-9, we hypothesized that a predictive genetic classifier ought to mirror both these features. In a panel of patient-derived melanoma xenografts10 (PDX), we noted that cells derived from the human tumor microenvironment (TME) that were co-grafted with the tumor cells were naturally replaced by murine cells after the first passage. Taking advantage of the XenofilteR11 algorithm we recently developed to deconvolute human from murine RNA sequence reads from PDX10, we obtained curated human melanoma tumor cell RNA reads. These expression signals were computationally subtracted from the total RNA profiles in bulk (tumor cell + TME) melanomas from patients. We thus derived one genetic signature that is purely tumor cell-intrinsic ("InTumor"), and one that comprises tumor cell-extrinsic RNA profiles ("ExTumor"). Here we report that the InTumor signature predicts patient response to anti-PD-1, but not anti-CTLA-4 treatment. This was validated in melanoma PDX and cell lines, which confirmed that InTumorLO tumors were effectively eliminated by adoptive cell transfer of T-Cell Receptor (TCR)-matched cytotoxic T cells, whereas InTumorHI melanomas were refractory and grew out as fast as tumors challenged with unmatched T cells. In contrast, the ExTumor signature predicts patient response to anti-CTLA-4 but not anti-PD-1. Most importantly, we used the InTumor and ExTumor signatures in conjunction to generate an ICB response quadrant, which predicts clinical benefit for five independent melanoma patient cohorts treated with either mono- or combination ICB. Specifically, these signatures enable identification of patients who have a much higher chance of responding to the combination treatment than to either monotherapy (p < 0.05), as well as patients who are likely to experience little benefit from receiving anti-CTLA-4 on top of anti-PD-1 (p < 0.05). These signatures may be clinically exploited to distinguish patients who need combined PD-1 + CTLA-4 blockade from those who are likely to benefit from either anti-CTLA-4 or anti-PD-1 monotherapy.

cancer biology↗

bioRxiv2020

LOSS OF UBIQUITIN LIGASE STUB1 AMPLIFIES IFNγ-R1/JAK1 SIGNALING AND SENSITIZES TUMORS TO IFNγ

Despite the success of immune checkpoint blockade (ICB) most patients fail to respond durably, in part owing to reduced interferon gamma (IFNγ) sensitivity. Thus, elevating tumor IFNγ-receptor 1 (IFNγ-R1) expression to enhance IFNγ-mediated cytotoxicity is of potential clinical interest. Here, we show that increased IFNγ-R1 expression sensitizes tumors to IFNγ-mediated killing. To unveil the largely undefined mechanism governing IFNγ-R1 expression, we performed a genome-wide CRISPR/Cas9 screen for suppressors of its cell surface abundance. We uncovered STUB1 as key mediator of proteasomal degradation of the IFNγ-R1/JAK1 complex. STUB1 inactivation amplified IFNγ signaling, thereby sensitizing to cytotoxic T cells, but also inducing PD-L1. STUB1 loss in a rational combination with PD-1 blockade strongly inhibited melanomas in vivo. Clinically corroborating these results, a STUB1-KO gene signature was strongly associated with anti-PD-1 response. These results uncover STUB1 as pivotal regulator of IFNγ tumor signaling and provide a rationale for its inhibition combined with anti-PD-1.Competing Interest StatementD.S.P. is co-founder, shareholder and advisor of Immagene B.V. M.A.L. is co-founder, shareholder and C.E.O. of Immagene B.V. The other authors report no competing financial interests. View Full Text

cancer biology↗

bioRxiv2019

Combined EGFR and ROCK inhibition in TNBC leads to cell death via impaired autophagic flux

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with very limited therapeutic options. We have recently shown that the combined inhibition of EGFR and ROCK in TNBC cells results in cell death, however, the underlying mechanisms remain unclear. To investigate this, here we applied a mass spectrometry-based proteomic approach to identify proteins altered upon single and combination treatments. Our proteomic data revealed autophagy as the major molecular mechanism implicated in the cells response to combinatorial treatment. In particular, we here show that EGFR inhibition by gefitinib treatment alone induces autophagy, a cellular recycling process that acts as a cytoprotective response for TNBC cells. However, combined inhibition of EGFR and ROCK leads to autophagy blockade and accumulation of autophagic vacuoles. Our data show impaired autophagosome clearance as a cause of antitumor activity. We propose that the inhibition of the autophagic flux upon combinatorial treatment is attributed to the major cytoskeletal changes induced upon ROCK inhibition, given the essential role the cytoskeleton plays throughout the various steps of the autophagy process.

cancer biology↗