Biology subjects

Freeman, G. J.

Publications and source records attributed to Freeman, G. J..

6 recordsLinked to original sources

bioRxiv2026

Targeting FGFR signaling overcomes therapeutic resistance and immune evasion in oncogenic PIK3CA-driven serous-like endometrial cancer

Serous endometrial cancer (SEC) is an aggressive subtype of endometrial cancer (EC) with poor prognosis and limited treatment options. Here, we developed a clinically relevant, immunocompetent serous-like mouse model incorporating oncogenic PIK3CA mutation, Trp53 loss, and MYC overexpression. Using this model together with human EC cell lines, patient-derived organoids (PDOs), xenografts, and patient datasets, we investigated mechanisms underlying resistance to PI3K-targeted therapy. Single-cell profiling reveals that FGFR1/2 upregulation associates with intrinsic resistance, whereas FGFR3 characterizes acquired resistance. Dual FGFR and PI3K inhibition produced superior tumor control compared with either agent alone. Mechanistically, FGFR signaling promotes immune evasion by downregulating MHC-I/HLA-mediated antigen presentation and enriching M2-type tumor-associated macrophages. FGFR inhibition reversed these changes and synergized with anti-PD-1 therapy to enhance antitumor immune responses and establish durable immune memory. Collectively, these findings identify FGFR signaling as a key driver of therapeutic resistance and immune escape in SEC and support FGFR-targeted combination strategies.

cancer biology↗

bioRxiv2024

Stratified analysis identifies HIF-2α as a therapeutic target for highly immune-infiltrated melanomas

While immune-checkpoint blockade (ICB) has revolutionized treatment of metastatic melanoma over the last decade, the identification of broadly applicable robust biomarkers has been challenging, driven in large part by the heterogeneity of ICB regimens and patient and tumor characteristics. To disentangle these features, we performed a standardized meta-analysis of eight cohorts of patients treated with anti-PD-1 (n=290), anti-CTLA-4 (n=175), and combination anti-PD-1/anti-CTLA-4 (n=51) with RNA sequencing of pre-treatment tumor and clinical annotations. Stratifying by immune-high vs -low tumors, we found that surprisingly, high immune infiltrate was a biomarker for response to combination ICB, but not anti-PD-1 alone. Additionally, hypoxia-related signatures were associated with non-response to anti-PD-1, but only amongst immune infiltrate-high melanomas. In a cohort of scRNA-seq of patients with metastatic melanoma, hypoxia also correlated with immunosuppression and changes in tumor-stromal communication in the tumor microenvironment (TME). Clinically actionable targets of hypoxia signaling were also uniquely expressed across different cell types. We focused on one such target, HIF-2, which was specifically upregulated in endothelial cells and fibroblasts but not in immune cells or tumor cells. HIF-2 inhibition, in combination with anti-PD-1, enhanced tumor growth control in pre-clinical models, but only in a more immune-infiltrated melanoma model. Our work demonstrates how careful stratification by clinical and molecular characteristics can be leveraged to derive meaningful biological insights and lead to the rational discovery of novel clinical targets for combination therapy.

cancer biology↗

bioRxiv2022

Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma

Immune checkpoint blockers (ICBs) have failed in all Phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following anti-PD1 antibody treatment that disrupts the blood-tumor-barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients. One-Sentence SummaryLosartan prevents immunotherapy-associated edema and enhances the outcome of immunotherapy in glioblastoma.

cancer biology↗

bioRxiv2021

Immune checkpoint expression on HIV-specific CD4+ T cells and response to their blockade are dependent on lineage and function

BackgroundAntigen-specific T cell impairment is observed in chronic infections. CD4+ T cells are diverse in phenotype and function; how their different lineages are impacted by inhibitory immune checkpoints (IC) is unknown. MethodsWe examined IC expression and function in HIV-specific CD4+ T cells of viremic individuals prior to ART initiation and persons with spontaneous or therapy-induced viral suppression. We investigated IC patterns associated with exhaustion-related transcription factors and chemokine receptors using cytokine-independent activation-induced marker assays. We determined effector functions representative of TFH, TH1 and TH17/TH22 using ultra-sensitive RNA flow cytometric fluorescence in situ hybridization (FISH), and their response to IC blockade. FindingsThe dysfunction-related transcription factor TOX was elevated in HIV-specific CD4+ T cells of viremic patients, and its expression was associated with lineage differentiation. We observed a hierarchy of PD-1, TIGIT and CD200 expression associated with both infection status and effector profile. In vitro responsiveness to PD-L1 blockade varied with defined CD4+ T cell functions rather than IC expression levels: frequencies of cells with TH1- and TH17/TH22-, but not TFH-related functions, increased. Response to PD-L1 blockade was strongest in viremic participants and reduced after ART initiation. InterpretationOur data highlight a polarization-specific regulation of IC expression and differing sensitivities of antigen-specific Thelper subsets to PD-1-mediated inhibition. This heterogeneity may direct ICB efficacy on CD4+ T cells in HIV infection. FundingThis work was supported by the National Institutes of Health, the Canadian Institutes for Health Research, the Canada Foundation for Innovation and the Fonds de Recherche du Quebec-Sante. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSCombination antiretroviral therapy (ART) is highly effective in controlling HIV but requires life-long medication due to the latent viral reservoir, and does not restore suppressive immune responses. In particular, there is no generation of effective HIV-specific T cell responses, which are thought to play an important role in controlling HIV in the rare individuals who can spontaneously control the virus. Inhibitory immune checkpoints (IC) such as PD-1 contribute to T cell dysfunction and failure to control viral infections, including HIV, and IC blockade (ICB) represents a potential adjuvant to ART through restoration of T cell functions. While most studies have focused on CD8+ T cells, increasing evidence shows that the remarkable impact of ICB therapy in a subset of cancer patients is enhanced by functional CD4+ T cell help, which can be directly affected by ICB. While effective virus-specific CD4+ T cell responses are also thought to be important for immune control of HIV, these cells are highly heterogenous. How IC expression and function differs across CD4+ T cell lineages and the consequences of this diversity for IC blockade (ICB) strategies are still poorly understood. Added value of this studyTo compare various stages of immune dysfunction, we examined people living with HIV (PLWH) with different levels of viral control pre-ART (including elite controllers who spontaneously control virus) and followed a cohort longitudinally post-ART. We used a panel of assays to characterize HIV-specific CD4+ T cell subsets, including activation-induced marker (AIM) assays and flow cytometric detection of mRNAs coding for a wider variety of HIV-specific CD4+ T cell functions than what is detected by standard procedures. Our experiments indicate a hierarchy of IC (PD-1, TIGIT, CD200) expression on blood HIV-specific CD4+ T cells that depends not only on the persons infection status but also on expression of lineage differentiation markers and effector functions representative of CD4+ T cell subsets critical for antiviral responses (TFH, TH1 and TH17/TH22 cells). This hierarchy was also present in the putatively functional cells of elite controllers. We characterized the expression of the dysfunction-related transcription factor TOX, and saw that its association with the key IC PD-1 in the setting of viremia varied across CD4+ T cell polarizations. Response to blockade of the PD-1 pathway resulted in increased antiviral and mucosal-protective functions, but did not affect TFH-related functions. Response to ICB was most prominent in viremic patients, and subdued but not fully abrogated in the setting of viral suppression. Implications of all available evidenceThese results highlight a previously unrecognized impact of ICB on mucosal immunity-related CD4+ functions, which are known to be depleted upon HIV infection and not restored by ART, and strong links between IC expression patterns and HIV-specific CD4+ T cell differentiation. The impact of ICB on CD4+ T cells in HIV infection has primarily been studied in the context of viral reservoir reactivation, which are preferentially harbored in IC+ cells. Our work emphasizes the importance of considering the differentiation profile of the virus-specific CD4+ T cells in studies of ICB blockade, as it may direct ICB efficacy in HIV infection. This data may also have implications for CD4+ T cell help in other infectious and non-infectious chronic human diseases.

immunology↗

bioRxiv2021

Functional inactivation of pulmonary MAIT cells following 5-OP-RU treatment of non-human primates

Targeting MAIT cells holds promise for the treatment of different diseases and infections. We previously showed that treatment of Mycobacterium tuberculosis infected mice with 5-OP-RU, a major antigen for MAIT cells, expands MAIT cells and enhances bacterial control. Here we treated M. tuberculosis infected rhesus macaques with 5-OP-RU intratracheally but found no clinical or microbiological benefit in M. tuberculosis infected macaques. In fact, after 5-OP-RU treatment MAIT cells did not expand, but rather upregulated PD-1 and lost the ability to produce multiple cytokines, a phenotype resembling T cell exhaustion. Furthermore, we show that vaccination of uninfected macaques with 5-OP-RU+CpG instillation into the lungs also drives MAIT cell dysfunction, and PD-1 blockade during vaccination partly prevents the loss of MAIT cell function without facilitating their expansion. Thus, in rhesus macaques MAIT cells are prone to the loss of effector functions rather than expansion after TCR stimulation in vivo, representing a significant barrier to therapeutically targeting these cells.

immunology↗

bioRxiv2020

PD-1 blockade exacerbates Mycobacterium tuberculosis infection in rhesus macaques

Boosting immune cell function by targeting the co-inhibitory receptor PD-1 may have applications in the treatment of chronic infections. Here we examine the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. Animals treated with PD-1 mAb developed worse disease and higher granuloma bacterial loads compared to isotype control treated monkeys. PD-1 blockade increased the number and functionality of granuloma Mtb-specific CD8 T cells. In contrast, Mtb-specific CD4 T cells in PD-1 treated macaques were not increased in number or function in granulomas, upregulated high levels of CTLA-4 and exhibited reduced intralesional trafficking in live imaging studies. In granulomas of PD-1 treated animals, multiple pro-inflammatory cytokines were elevated, and more cytokines correlated with bacterial loads, leading to the identification of a role for caspase 1 in the exacerbation of tuberculosis after PD-1 blockade. Lastly, increased Mtb bacterial loads after PD-1 blockade were found to associate with the composition of the intestinal microbiota prior to infection in individual macaques. Therefore, PD-1-mediated co-inhibition is required for control of Mtb infection in macaques, perhaps due to its role in dampening detrimental inflammation as well as allowing for normal CD4 T cell responses.

immunology↗