Biology subjects

Merghoub, T.

Publications and source records attributed to Merghoub, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Mutant p53 Directs PARP to Regulate Replication Stress and Drive Breast Cancer Metastasis

TP53 mutations occur in 80-90% of triple-negative breast cancers (TNBCs) and drive genomic instability and metastatic progression. Poly (ADP-ribose) polymerase (PARP) is critical for DNA repair and replication fork stability. How oncogenic signaling influences PARP function to sustain proliferation during replication stress remains unclear. Mutant p53 (mtp53) R273H associates tightly with chromatin, forms complexes with PARP, and enhances PARP recruitment to replication forks [1-3]. The C-terminal region of mtp53 mediates mtp53-PARP and mtp53-Poly (ADP-ribose) (PAR) interactions that facilitate S phase progression [4, 5]. The PARP inhibitor talazoparib (TAL) combined with the alkylating agent temozolomide (TMZ) produces synergistic cytotoxicity selectively in mtp53, but not wild-type p53 (wtp53), breast cancer cells and organoids. Herein we evaluated the mechanism of mtp53-associated cell death and tested if this could translate to a preclinical xenograft model. We found that TMZ+TAL treatment induced elevated cleaved PARP and {gamma}H2AX and reduced the metastasis-promoting oncoprotein MDMX. In orthotopic xenografts expressing mtp53 R273H, but not wtp53, combination therapy significantly decreased circulating tumor cells (CTCs) and lung metastases. Transcriptomic profiling of tumors from combination treated animals demonstrated downregulation of MDMX, VEGF, and NF-{kappa}B, consistent with the observed suppression of CTCs and lung metastasis, and increased {gamma}H2AX, indicative of replication stress in mtp53 xenografts. Inhibition of metastasis was also observed in mtp53 R273H WHIM25 and p53-undetectable WHIM6 TNBC patient-derived xenografts (PDX). The mtp53 C-terminal domain (347-393) demonstrated a critical tumor promoting function, as CRISPR-mediated deletion impaired replication fork progression, tumor growth, and metastatic dissemination. DNA fiber combing showed that expression of full-length mtp53 R273H, but not C-terminal deleted {Delta}347-393, supported sustained single-stranded DNA gaps (ssGAPs) following Poly (ADP-ribose) glycohydrolase (PARG) inhibition. These findings support that mtp53 uses C-terminal amino acids to exploit PARP to enable replication stress adaptation and that mtp53 is a predictive biomarker for combined PARP inhibitor and DNA damaging therapies targeting TNBC. Significance statementTP53 mutations are the most common genetic alterations in TNBC and a major driver of replication stress and metastasis. This study shows that missense mutant p53 uses C-terminal amino acids to reprogram PARP activity to maintain tumor cell survival under replication stress. We demonstrate that p53 status governs the response to combined PARP inhibitor (PARPi) and DNA-damaging chemotherapy, establishing an additional molecular basis beyond BRCA1 mutations for treating TNBC with PARPi therapy. These findings reveal a previously unrecognized mechanism by which the mutant p53-PARP axis enables replication stress tolerance and drives cancer metastasis. We show mutation of p53 in TNBC provides an additional biomarker-guided framework to improve PARPi therapeutic outcomes.

cancer biology↗

bioRxiv2025

Human myelocyte and metamyelocyte-stage neutrophils suppress tumor immunity and promote cancer progression

Tumor-infiltrating neutrophils (TINs) are highly heterogeneous and mostly immunosuppressive in the tumor immune microenvironment (TIME). Current biomarkers of TINs and treatment strategies targeting TINs have not yielded optimal responses in patients across cancer types. Here, we separated human and mouse neutrophils into three developmental stages, including promyelocyte (PM), myelocyte & metamyelocyte (MC & MM), and banded & segmented (BD & SC) neutrophils. Based on this separation, we observed the predominance of human but not mouse MC & MM-stage neutrophils in bone marrow (BM), which exhibit potent immunosuppressive and tumor-promoting properties. MCs & MMs also occupy the majority of TINs among patients with 17 cancer types. Moreover, through the creation of a NCG-Gfi1-/- human immune system (HIS) mouse model, which supports efficient reconstitution of human TIN, we found a significant increase of BM MCs & MMs in tumor-bearing mice. By comparing the scRNA-seq analysis results of human neutrophils from both BM and tumors, we found CD63 and Galectin-3 distinguish MC & MM from neutrophil populations in cancer patients. Furthermore, we proposed a strategy with Flt3L to specifically induce the trans-differentiation of MCs & MMs into monocytic cells, and trigger tumor control in NCG-Gfi1-/- HIS mice. Thus, our findings establish an essential role of human MC & MM-stage neutrophils in promoting cancer progression, and suggest their potential as targets for developing potential biomarkers and immunotherapies for cancer.

immunology↗

bioRxiv2025

A pathogenic subpopulation of human glioma associated macrophages linked to glioma progression

Malignant gliomas follow two distinct natural histories: de novo high grade tumors such as glioblastoma, or lower grade tumors with a propensity to transform into high grade disease. Despite differences in tumor genotype, both entities converge on a common histologically aggressive phenotype, and the basis for this progression is unknown. Glioma associated macrophages (GAM) have been implicated in this process, however GAMs are ontologically and transcriptionally diverse, rendering isolation of pathogenic subpopulations challenging. Since macrophage contextual gene programs are orchestrated by transcription factors acting on cis-acting promoters and enhancers in gene regulatory networks (GRN), we hypothesized that functional populations of GAMs can be resolved through GRN inference. Here we show via parallel single cell RNA and ATAC sequencing that a subpopulation of human GAMs can be defined by a GRN centered around the Activator Protein-1 transcription factor FOSL2 preferentially enriched in high grade tumors. Using this GRN we nominate ANXA1 and HMOX1 as surrogate cell surface markers for activation, thus permitting prospective isolation and functional validation in human GAMs. These cells, termed malignancy associated GAMs (mGAMs) are pro-invasive, pro-angiogenic, pro-proliferative, possess intact antigen presentation but skew T-cells towards a CD4+FOXP3+ phenotype under hypoxia. Ontologically, mGAMs share somatic mitochondrial mutations with peripheral blood monocytes, and their presence correlates with high grade disease irrespective of underlying tumor mutation status. Furthermore, spatio-temporally mGAMs occupy distinct metabolic niches; mGAMs directly induce proliferation and mesenchymal transition of low grade glioma cells and accelerate tumor growth in vivo upon co-culture. Finally mGAMs are preferentially enriched in patients with newly transformed regions in human gliomas, supporting the view that mGAMs play a pivotal role in glioma progression and may represent a plausible therapeutic target in human high-grade glioma.

cancer biology↗

bioRxiv2024

TAD hierarchy restricts poised LTR activation and loss of TAD hierarchy promotes LTR co-option in cancer

Transposable elements (TEs) are abundant in the human genome, and they provide the sources for genetic and functional diversity. The regulation of TEs expression and their functional consequences in physiological conditions and cancer development remain to be fully elucidated. Previous studies suggested TEs are repressed by DNA methylation and chromatin modifications. The effect of 3D chromatin topology on TE regulation remains elusive. Here, by integrating transcriptome and 3D genome architecture studies, we showed that haploinsufficient loss of NIPBL selectively activates alternative promoters at the long terminal repeats (LTRs) of the TE subclasses. This activation occurs through the reorganization of topologically associating domain (TAD) hierarchical structures and recruitment of proximal enhancers. These observations indicate that TAD hierarchy restricts transcriptional activation of LTRs that already possess open chromatin features. In cancer, perturbation of the hierarchical chromatin topology can lead to co-option of LTRs as functional alternative promoters in a context-dependent manner and drive aberrant transcriptional activation of novel oncogenes and other divergent transcripts. These data uncovered a new layer of regulatory mechanism of TE expression beyond DNA and chromatin modification in human genome. They also posit the TAD hierarchy dysregulation as a novel mechanism for alternative promoter-mediated oncogene activation and transcriptional diversity in cancer, which may be exploited therapeutically.

cancer biology↗

bioRxiv2022

IL-12-expressing highly immunogenic recombinant modified vaccinia virus Ankara reprograms tumor-infiltrating myeloid cells to overcome immune resistance

Resistance to immune checkpoint blockade (ICB) remains a major obstacle in cancer immunotherapy. We rationally engineered a second-generation recombinant Modified Vaccinia virus Ankara (MQ833) by deleting viral immune-evasion genes (E3L, E5R, WR199) and incorporating Flt3L, OX40L, and matrix-anchored IL12. Intratumoral MQ833 elicited robust tumor regressions across multiple models, including ICB-resistant and MHC-I-deficient tumors. Single-cell RNA sequencing revealed extensive remodeling of the tumor microenvironment, characterized by neutrophil and monocyte recruitment and activation, M2 macrophage depletion, M1 polarization, and effector T-cell differentiation and proliferation. Conditional Ifnar1 knockout mice demonstrated that MQ833 efficacy requires type I interferon signaling in neutrophils, macrophages/monocytes, and T cells. Moreover, Nos2 deficiency impaired therapeutic efficacy, confirming iNOS myeloid cells as key effectors. Together, these findings show that MQ833 activates innate-adaptive IFN cross-talk to reprogram myeloid and T cells, defining a rationally designed viral immunotherapy capable of overcoming ICB resistance. GRAPHIC ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/509429v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@496f37org.highwire.dtl.DTLVardef@124bd78org.highwire.dtl.DTLVardef@1134528org.highwire.dtl.DTLVardef@216da6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

An Immune Cell Recirculation-Enabled Microfluidic Array to Study Dynamic Immunotherapeutic Activity in Recapitulated Tumor Microenvironment

The efficacy of immunotherapeutic treatment protocols to enable immune cell mediated treatment of cancer is significantly modulated in the presence of tumor microenvironment (TME) which is a key factor in providing both a physical barrier and immunosuppressive stimuli. Herein, we developed a recirculating, high-throughput microfluidic cell array to capture these crucial players - cytotoxic T cells in circulation, endothelium, and tumor stroma. The system consisted of a three-layered cell array spatially emulating TME, with T cell circulation sustained via fluidic recirculating circuits. This allowed us to study the dynamic TME/circulation system and cancer cell response thereof. The system further revealed that tumor endothelium exhibited a hindrance to T cell infiltration into the breast cancer tumor compartment, which was alleviated when treated with anti-human PD-L1 antibody. The other key stromal component, cancer associated fibroblasts, further attenuated T cell infiltration, and led to reduced apoptosis activity in cancer cells. These results confirm the capability of our tumor-on-a-chip system to recapitulate some key immune cell interactions with the reconstructed TME, along with demon-strating as the feasibility of using this system for high-throughput cancer immunotherapeutic screening.

bioengineering↗

bioRxiv2022

Leveraging CD39 To Identify Tumor-Reactive CD8 T cells In Human Lung Cancer

The repertoire of tumor-infiltrating lymphocytes (TILs) can be vast, and many of these TILs are not endowed with tumor reactivity. While a number of reports have shown that tumor-reactive TILs express CD39, few reports have demonstrated that conversely, CD39 can be leveraged to serve as a proxy of tumor-reactive CD8 T cells. Using single-cell CITE/RNA/TCRseq, we show that CD39+ CD8 T cells in human lung cancers demonstrate transcriptional and proteomic features of exhaustion, tumor reactivity, and clonal expansion. Moreover, TCR cloning revealed that CD39 enriched for tumor-reactive CD8 T cell clones. Flow cytometry of 440 lung cancer specimens revealed that CD39 level on CD8 T cells is only weakly correlated with tumoral features that currently guide lung cancer therapy, such as histology, driver mutation, PD-L1 and tumor mutation burden. PD-1 axis blockade, but not cytotoxic chemotherapy, increased intratumoral CD39+ CD8 T cells. CD39 correlated with PD-1 expression on CD8 T cells and high pre-treatment/early-on-treatment levels were associated with improved clinical outcomes, but not immune-related adverse events, from immune checkpoint blockade therapy. This comprehensive profiling of the clinical, pathological and molecular features highlights the utility of CD39 as a proxy for tumor-reactive CD8 T cells in human lung cancer.

immunology↗

bioRxiv2021

A nuclear cAMP microdomain suppresses tumor growth by Hippo pathway inactivation

cAMP signaling pathways are critical for both oncogenesis and tumor suppression. cAMP signaling is localized to multiple spatially distinct microdomains, but the role of cAMP microdomains in cancer cell biology is poorly understood. We developed a tunable genetic system that allows us to activate cAMP signaling in specific microdomains. We uncovered a previously unappreciated nuclear cAMP microdomain that functionally activates a tumor suppressive pathway in a broad range of cancers by inhibiting YAP, a key effector protein of the Hippo pathway, inside the nucleus. We show that nuclear cAMP induces a LATS-dependent pathway leading to phosphorylation of nuclear YAP solely at serine 397, export of YAP from the nucleus, without YAP protein degradation. Thus, nuclear cAMP inhibition of nuclear YAP is distinct from other known mechanisms of Hippo regulation. Pharmacologic targeting of specific cAMP microdomains remains an untapped therapeutic approach for cancer, and since Hippo pathway deregulation can lead to oncogenesis and chemotherapeutic resistance, drugs directed at the nuclear cAMP microdomain may provide new avenues for the treatment of cancer.

cancer biology↗

bioRxiv2021

Intratumoral delivery of engineered recombinant modified vaccinia virus Ankara express-ing Flt3L and OX40L generates potent antitumor immunity through activating the cGAS/STING pathway and depleting tumor-infiltrating regulatory T cells

Intratumoral (IT) delivery of immune-activating viruses can serve as an important strategy to turn "cold" tumors into "hot" tumors, resulting in overcoming resistance to immune checkpoint blockade (ICB). Modified vaccinia virus Ankara (MVA) is a highly attenuated, non-replicative vaccinia virus that has a long history of human use. Here we report that IT recombinant MVA (rMVA), lacking E5R encoding an inhibitor of the DNA sensor cyclic GMP-AMP synthase (cGAS), expressing a dendritic cell growth factor, Fms-like tyrosine kinase 3 ligand (Flt3L), and a T cell co-stimulator, OX40L, generates strong antitumor immunity, which is dependent on CD8+ T cells, the cGAS/STING-mediated cytosolic DNA-sensing pathway, and STAT1/STAT2-mediated type I IFN signaling. Remarkably, IT rMVA depletes OX40hi regulatory T cells via OX40L/OX40 interaction and IFNAR signaling. Taken together, our study provides a proof-of-concept for improving MVA-based cancer immunotherapy, through modulation of both innate and adaptive immunity. One Sentence SummaryIntratumoral delivery of recombinant MVA for cancer immunotherapy

immunology↗

bioRxiv2021

Regional and clonal T cell dynamics at single cell resolution in immune checkpoint blockade

Paired T cell receptor and RNA single cell sequencing (scTCR/RNA-seq) has allowed for enhanced resolution of clonal T cell dynamics in cancer. Here, we report a scTCR/RNA-seq dataset of 162,062 single T cells from 31 tissue regions, including tumor, adjacent normal tissues, and lymph nodes (LN), from three patients who underwent resections for progressing lung cancers after immune checkpoint blockade (ICB). We found marked regional heterogeneity in tumor persistence that was associated with heterogeneity in CD4 and CD8 T cell phenotypes; regions with persistent cancer cells were enriched for follicular helper CD4 T cells (TFH), regulatory T cells (Treg), and exhausted CD8 T cells. Clonal analysis demonstrated that highly-expanded T cell clones were predominantly of the CD8 subtype, were ubiquitously present across all sampled regions, found in the peripheral circulation, and expressed gene signatures of large and dual-expanded clones that have been predictive of response to ICB. Longitudinal tracking of CD8 T cell clones in the peripheral blood revealed that the persistence of ubiquitous CD8 T cell clones, as well as phenotypically distinct clones with tumor-reactive features, correlated with systemic tumor control. Finally, tracking CD8 T cell clones across tissues revealed the presence of TCF-1+ precursor exhausted CD8 T cells in tumor draining LNs that were clonally linked to expanded exhausted CD8 T cells in tumors. Altogether, this comprehensive scTCR/RNA-seq dataset with regional, longitudinal, and clonal resolution provides fundamental insights into the tissue distribution, persistence, and differentiation trajectories of ICB-responsive T cells that underlie clinical responses to ICB.

immunology↗

bioRxiv2021

Heat-inactivated modified vaccinia virus Ankara boosts Th1-biased cellular and humoral immune responses as a vaccine adjuvant by activating the STING-mediated cytosolic DNA-sensing pathway

BackgroundProtein or peptide-based subunit vaccines are promising platforms for combating human cancers and infectious diseases. However, one primary concern regarding subunit vaccines is the relatively weak immune responses induced by proteins or peptides. Therefore, developing novel and effective vaccine adjuvants is critical for the success of subunit vaccines. Modified vaccinia virus (MVA) is a safe and effective vaccine against smallpox and monkeypox. In this study, we explored the potential of heat-inactivated MVA (heat-iMVA) as a novel vaccine adjuvant. MethodsWe co-administered heat-iMVA with a model antigen, chicken ovalbumin (OVA), either intramuscularly or subcutaneously twice, two weeks apart, and analyzed anti-OVA specific CD8+ and CD4+ T cells in the spleens and skin draining lymph nodes (dLNs) and serum anti-OVA IgG1 and IgG2c antibodies. We also compared the adjuvanticity of heat-iMVA with several known vaccine adjuvants, including complete Freunds adjuvant (CFA) and AddaVax, an MF59-like preclinical grade nano-emulsion. In addition, we tested whether co-administration of heat-iMVA plus tumor neoantigen peptides or irradiated tumor cells improves antitumor efficacy in a B16-F10 therapeutic vaccination model. Using Stimulator of Interferon Genes (STING) or Batf3-deficient mice, we evaluated the contribution of the STING pathway and Batf3-dependent CD103+/CD8 DCs in heat-iMVA-induced immunity. ResultsCo-administration of protein- or peptide-based immunogens with heat-iMVA dramatically enhances Th1-biased cellular and humoral immune responses. This adjuvant effect of heat-iMVA is dependent on the STING-mediated cytosolic DNA-sensing pathway, and the antigen-specific CD8+ T cell response requires Batf3-dependent CD103+/CD8+ dendritic cells (DCs). Heat-iMVA infection of bone marrow-derived DCs (BMDCs) promoted antigen cross-presentation, whereas live MVA infection did not. RNA-seq analyses revealed that heat-iMVA is a more potent activator of the STING pathway than live MVA. Additionally, combining tumor neoantigen peptides or irradiated tumor cells with heat-iMVA delayed tumor growth and extended the median survival in B16-F10 therapeutic vaccination models. ConclusionsHeat-iMVA induces type I interferon (IFN) production and antigen cross-presentation via a STING-dependent mechanism in DCs. Co-administration of heat-iMVA with peptide antigen generates strong Th1-biased cellular and humoral immunity. Collectively, our results demonstrate that heat-iMVA is a safe and potent vaccine adjuvant.

immunology↗

bioRxiv2021

Clinical and molecular features of acquired resistance to immunotherapy in non-small cell lung cancer

Although cancer immunotherapy with PD-(L)1 blockade is now routine treatment for patients with lung cancer, remarkably little is known about acquired resistance. We examined 1,201 patients with NSCLC treated with PD-(L)1 blockade to clinically characterize acquired resistance, finding it to be common (occurring in more than 60% of initial responders), with persistent but diminishing risk over time, and with distinct metastatic and survival patterns compared to primary resistance. To examine the molecular phenotype and potential mechanisms of acquired resistance, we performed whole transcriptome and exome tumor profiling in a subset of NSCLC patients (n=29) with acquired resistance. Systematic immunogenomic analysis revealed that tumors with acquired resistance generally had enriched signals of inflammation (including IFN{gamma} signaling and inferred CD8+ T cells) and could be separated into IFN{gamma} upregulated and stable subsets. IFN{gamma} upregulated tumors had putative routes of resistance with signatures of dysfunctional interferon signaling and mutations in antigen presentation genes. Transcriptomic profiling of cancer cells from a murine model of acquired resistance to PD-(L)1 blockade also showed evidence of dysfunctional interferon signaling and acquired insensitivity to in vitro interferon gamma treatment. In summary, we characterized clinical and molecular features of acquired resistance to PD-(L)1 blockade in NSCLC and found evidence of ongoing but dysfunctional IFN response. The persistently inflamed, rather than excluded or deserted, tumor microenvironment of acquired resistance informs therapeutic strategies to effectively reprogram and reverse acquired resistance.

cancer biology↗

bioRxiv2021

Elucidating Mechanisms of Antitumor Immunity Mediated by Live Oncolytic Vaccinia and Heat-Inactivated Vaccinia

BackgroundViral-based immunotherapy has the potential to overcome resistance to immune checkpoint blockade (ICB) and to fill the unmet needs of many cancer patients. Oncolytic viruses (OVs) are defined as engineered or naturally occurring viruses that selectively replicate in and kill cancer cells. OVs also induce antitumor immunity. The purpose of this study is to compare the antitumor effects of live OV-GM expressing murine granulocyte-macrophage colony-stimulating factor (mGM-CSF) versus inactivated OV-GM and elucidate the underlying immunological mechanisms. MethodsIn this study, we engineered a replication-competent, oncolytic vaccinia virus (OV-GM) by inserting a murine GM-CSF gene into the thymidine kinase (TK) locus of a mutant vaccinia E3L{Delta}83N, which lacks the Z-DNA-binding domain of vaccinia virulence factor E3. We compared the antitumor effects of intratumoral (IT) delivery of live OV-GM vs. heat-inactivated OV-GM (heat-iOV-GM) in a murine B16-F10 melanoma bilateral implantation model. ResultsHeat-iOV-GM infection of dendritic cells (DCs) and tumor cells in vitro induces type I IFN and pro inflammatory cytokines and chemokines, whereas live OV-GM does not. IT live OV-GM is less effective in generating systemic antitumor immunity compared with heat-iOV-GM. Similar to heat-iOV-GM, the antitumor effects of live OV-GM also require Batf3-dependent CD103+ dendritic cells. IT heat-iOV-GM induces higher numbers of infiltrating activated CD8+ and CD4+ T cells as well as higher levels of type I IFN, proinflammatory cytokines, and chemokines in the distant non-injected tumors, which is dependent on CD8+ T cells. When combined with systemic delivery of ICB, IT heat-iOV-GM is more effective in eradicating tumors compared with live OV-GM. ConclusionsTumor lysis induced by the replication of oncolytic DNA viruses has a limited effect on the generation of systemic antitumor immunity. The activation of Batf3-dependent CD103+ DCs is critical for antitumor effects induced by both live OV-GM and heat-iOV-GM. Heat-iOV-GM is more potent than live OV-GM in the induction of innate and adaptive immunity in both the injected and distant non-injected tumors. We propose that evaluations of both innate and adaptive immunity induced by IT oncolytic viral immunotherapy at injected and non-injected tumors should be included as potential biomarkers.

immunology↗

bioRxiv2020

Anatomic position determines oncogenic specificity in melanoma

Oncogenic alterations to DNA are not transforming in all cellular contexts1,2. This may be due to pre-existing transcriptional programs in the cell of origin. Here, we define anatomic position as a major determinant of why cells respond to specific oncogenes. Cutaneous melanoma arises throughout the body, whereas the acral subtype arises on the palms of the hands, soles of the feet, or under the nails3. We sequenced the DNA of cutaneous and acral melanomas from a large cohort of human patients and found a specific enrichment for BRAF mutations in cutaneous melanoma but CRKL amplifications in acral melanoma. We modeled these changes in transgenic zebrafish models and found that CRKL-driven tumors predominantly formed in the fins of the fish. The fins are the evolutionary precursors to tetrapod limbs, indicating that melanocytes in these acral locations may be uniquely susceptible to CRKL. RNA profiling of these fin/limb melanocytes, compared to body melanocytes, revealed a positional identity gene program typified by posterior HOX13 genes. This positional gene program synergized with CRKL to drive tumors at acral sites. Abrogation of this CRKL-driven program eliminated the anatomic specificity of acral melanoma. These data suggest that the anatomic position of the cell of origin endows it with a unique transcriptional state that makes it susceptible to only certain oncogenic insults.

cancer biology↗

bioRxiv2020

Oxidized Lipids and CD36-Mediated Lipid Peroxidation in CD8 T Cells Suppress Anti-Tumor Immune Responses

T cell metabolic fitness plays a pivotal role in anti-tumor immunity and metabolic deregulation causes T cell dysfunction (i.e., exhaustion) in cancer. We identify that the scavenger receptor CD36 limits anti-tumor CD8+ T cell effector functions through lipid peroxidation. In murine tumors, oxidized phospholipids (OxPLs) were highly abundant and CD8+ TILs increased uptake and accumulation of lipids and lipid peroxidation. Functionally exhausted CD8+ TILs substantially increased CD36 expression and CD36-deficient CD8+ TILs had more robust anti-tumor activity and cytokine production than wild-type cells. We further show that CD36 promotes uptake of oxidized low-density lipoproteins (OxLDL) and induces lipid peroxidation in CD8+ TILs, and OxLDL inhibits CD8+ T cell functions in a CD36-dependent manner. Moreover, glutathione peroxidase 4 (GPX4) over-expression lowers lipid peroxidation and restores functionalities in CD8+ TILs. These results define a key role for an oxidized lipid-CD36 axis in promoting intratumoral CD8+ T cell dysfunction.

immunology↗

bioRxiv2019

Escape from nonsense mediated decay associates with anti-tumor immunogenicity

Frameshift insertion/deletions (fs-indels) are an infrequent but potentially highly immunogenic mutation subtype. Although fs-indel transcripts are susceptible to degradation through the non-sense mediated decay (NMD) pathway, we hypothesise that some fs-indels escape degradation and lead to an increased abundance of tumor specific neoantigens, that are highly distinct from self. We analysed matched DNA and RNA sequencing data from TCGA, and five separate melanoma cohorts treated with immunotherapy. Using allele-specific expression analysis we show that expressed fs-indels were enriched in genomic positions predicted to escape NMD, and associated with higher protein expression, consistent with degradation escape ("NMD-escape"). Across four independent cohorts, fs-indel NMD-escape mutations were found to be significantly associated with clinical benefit to checkpoint inhibitor (CPI) therapy (Pmeta=0.0039), a stronger association than either nsSNV (Pmeta=0.073) or fs-indel (Pmeta=0.064) count. NMD-escape mutations were additionally shown to have independent predictive power in the "low-TMB" setting, and may serve as a biomarker to rescue patients judged ineligible for CPI based on overall TMB, but still with a high chance of response (low-TMB cohort: NMD-escape-positive % clinical benefit=53%, NMD-escape-negative % clinical benefit=16%, P=0.0098). Furthermore, in an adoptive cell therapy (ACT) treated cohort, NMD-escape mutation count was the most significant biomarker associated with clinical benefit (P=0.021). Analysis of functional T-cell reactivity screens from recent personalized vaccine and CPI studies shows direct evidence of fs-indel derived neoantigens eliciting patient anti-tumor immune response (n=15). We additionally observe a subset of fs-indel mutations, with highly elongated neo open reading frames, which are found to be significantly enriched for immunogenic reactivity in these patient studies (P=0.0032). Finally, consistent with the potency of NMD-escape derived neo-antigens and ongoing immune-editing, NMD-escape fs-indels appear to be under negative selective pressure in untreated TCGA cases. Given the strongly immunogenic potential, and relatively rare nature of NMD-escape fs-indels, these alterations may be attractive candidates in immunotherapy biomarker optimisation and neoantigen ACT or vaccine strategies.

cancer biology↗

bioRxiv2017

Immune-active microenvironment in small cell carcinoma of the ovary, hypercalcemic type: rationale for immune checkpoint blockade

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a highly aggressive monogenic cancer driven by SMARCA4 mutations. Here, we report responses to anti-PD1 immunotherapy in four patients and characterize the immune landscape of SCCOHT tumors using quantitative immunofluorescence and gene expression profiling. Unexpectedly for a low mutation burden cancer, the majority of the tumors demonstrated PD-L1 expression with strong associated T cell infiltration. PD-L1 expression was detected in both tumor and stromal cells, with macrophages being the most abundant PD-L1 positive cells in some tumors. Transcriptional profiling revealed increased expression of genes related to Th1 and cytotoxic cell function in PD-L1 high tumors, suggesting that PD-L1 acts as a pathway of adaptive immune resistance in SCCOHT. These findings suggest that although SCCOHT are low-mutational-burden tumors, their immunogenic microenvironment that resembles the landscape of tumors that respond well to treatment with PD-1/PD-L1 blockade.

cancer biology↗

bioRxiv2016

Somatic Mutations and Neoepitope Homology in Melanomas Treated with CTLA-4 Blockade

Immune checkpoint inhibitors are promising treatments for patients with a variety of malignancies. Toward understanding the determinants of response to immune checkpoint inhibitors, it was previously demonstrated that somatic mutation burden is associated with benefit and a hypothesis was posited that neoantigen homology to pathogens may in part explain the link between somatic mutations and response. To further examine this hypothesis, we reanalyzed cancer exome data obtained from a previously published study of 64 melanoma patients treated with CTLA-4 blockade and a new dataset of RNA-Seq data from 24 of those patients. We found that the predictive accuracy does not increase as analysis narrows from somatic mutation burden to predicted MHC Class I neoantigens, expressed neoantigens, or homology to pathogens. Further, the association between somatic mutation burden and response is only found when examining samples obtained prior to treatment. Neoantigen and expressed neoantigen burden are also associated with response, but neither is more predictive than somatic mutation burden. Neither the previously-described tetrapeptide signature nor an updated method to evaluate neoepitope homology to pathogens were more predictive than mutation burden.

cancer biology↗