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Hyperactivation of the PI3K pathway has been reported to correlate with resistance to immune checkpoint blockade therapy (ICB) in melanoma, highlighting the therapeutic potential of combining PI3K inhibition (PI3Ki) with ICB. To maximize the clinical benefit of PI3Ki-based immune oncology (IO) combination, we characterized the role of PI3K isoforms in tumor and T cells and determined the immunological impacts of PI3Ki alone or in combination with ICB. Inhibitions of PI3K were achieved by either genetic knockdown (KD) or the bioactive compound in PTEN-present (B16/MC38), PTEN-absent (BP/D4M) tumor cell lines, and CD8+ T cells (Pmel-1). Following PI3Ki, we determined the activation status of the PI3K pathway (p-AKT level), transcriptional profile, and cellular function of these cells. We found both in vitro KD and pharmacological inhibition of either PI3Kα or PI3Kβ displayed a dramatic reduction of the PI3K pathway in tumor cells but moderate or no reduction in T cells, whereas the PI3K pathway significantly decreased in T cells with PI3Kγ or PI3Kδ inhibition. KD of PI3Kα or β isoforms drastically sensitized both D4M and MC38 tumors to αPD1 in vivo. We also observed that only PI3Kγ or PI3Kδ inhibition profoundly suppressed cytokine production and cytotoxicity of CD8+ T cell, suggesting that PI3Kα or PI3Kβ isoform inhibition can achieve tumor specific PI3Ki with limited impacts on T cell function. Furthermore, we used multiple syngeneic melanoma models to determine whether PI3K isoform inhibition can synergize the antitumor activity of ICB in vivo. In PTEN-present tumors, BYL719 (BYL, a PI3Kα inhibitor) synergized with αPD1 to delay tumor growth and extend survival (median survival of MC38-bearing mice in control (Ctrl), BYL, αPD1, and combination (Comb) groups: 30, 36, 33, and >45 respectively; p Citation Format: Ritu Bohat, Xiaofang Liang, Chunyu Xu, Yitao Tang, Jiakai Hou, Nicholas A. Egan, Leilei Shi, Ashley Guerrero, Roshni Jaffery, Elizabeth M. Burton, Han Liang, Hussein Tawbi, Michael A. Davies, Weiyi Peng. Targeting PI3K isoforms to improve the effectiveness of T cell mediated immunotherapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4444.

BackgroundDespite approval of immunotherapy for wide ranges of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response which is partially attributed to immunosuppression co-opted by tumor cells. However, it is challenging to utilize conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in cancer patients.MethodsIn this study, an unbiased integrative strategy were designed to leverage the complementary strength of in vitro functional genomic screens and multi-omics clinical dataset to assess roles of individual tumor-intrinsic factors in regulating T cell tumor infiltration and T cell-mediated tumor killing, the two most important rate-limiting steps of cancer immunotherapy. Initially, a genome-wide CRISPR-Cas9 screening system using paired murine tumors and tumor-reactive T cells was employed to globally screen tumor intrinsic factors modulating the tumor sensitivity to T cell-mediated killing. Then, findings from the screening were integrated with the bioinformatics analysis of clinical datasets to further evaluate roles of each tumor intrinsic factor in governing antitumor immunity.ResultsThe integrative analysis successfully identified several novel tumor intrinsic factors as effectors of immune resistance, but also demonstrated distinct roles of these factors in controlling immune cell trafficking and tumor sensitivity to T cell-mediated killing. Among these factors, candidates controlling both rate-limiting steps of T cell tumor infiltration and T cell-mediated tumor killing were termed as ”Dual immune resistance regulators” and the remaining factors whose expression levels were not associated with tumor immune infiltration were termed as ”Cytotoxicity resistance regulators”. By selecting PRMT1 and RIPK1 as the representatives of these two groups respectively, we confirmed that genetically depletion of PRMT1 and RIPK1 sensitized tumors to T-cell mediated killing via two independent experimental approaches. Furthermore, inhibiting Prmt1 or Ripk1 sensitizes tumors to cancer immunotherapy, such as anti-PD-1 and anti-OX40 treatments (Tumor size (mm2) on day 21 after tumor inoculation: for anti-PD-1 treatment, Ctrl 84.05±23.10, PRMT1 KO 7.30±7.81, RIPK1 KO 2.03±4.96; for anti-OX40 treatment, Ctrl 81.04±7.72, PRMT1 KO 55.80±15.74, RIPK1 KO 38.78±14.06) and extended the survival of tumor-bearing mice. Moreover, by using a RIPK1-specific inhibitor, GSK2982772, we demonstrated that targeting cytotoxicity resistance regulators could enhance the antitumor activity of T cell-based cancer immunotherapy, despite limited impact on T cell tumor infiltration.ConclusionsCollectively, our data not only demonstrate distinct immunoregulatory roles and therapeutic potentials of PRMT1 and RIPK1 in T cell-mediated antitumor activity, but also provide a rich resource of novel targets for rational immuno-oncology combinations.

BackgroundDespite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response. These failures may be attributed to immunosuppressive mechanisms co-opted by tumor cells. However, it is challenging to use conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in patients with cancer.MethodsTo identify immunosuppressive mechanisms in non-responders to cancer immunotherapy in an unbiased manner, we performed genome-wide CRISPR immune screens and integrated our results with multi-omics clinical data to evaluate the role of tumor intrinsic factors in regulating two rate-limiting steps of cancer immunotherapy, namely, T cell tumor infiltration and T cell-mediated tumor killing.ResultsOur studies revealed two distinct types of immune resistance regulators and demonstrated their potential as therapeutic targets to improve the efficacy of immunotherapy. Among them, PRMT1 and RIPK1 were identified as a dual immune resistance regulator and a cytotoxicity resistance regulator, respectively. Although the magnitude varied between different types of immunotherapy, genetically targeting PRMT1 and RIPK1 sensitized tumors to T-cell killing and anti-PD-1/OX40 treatment. Interestingly, a RIPK1-specific inhibitor enhanced the antitumor activity of T cell-based and anti-OX40 therapy, despite limited impact on T cell tumor infiltration.ConclusionsCollectively, the data provide a rich resource of novel targets for rational immuno-oncology combinations.

Marek's disease (MD) is an alphaherpesvirus (Marek's disease virus, MDV)-induced pathology of chickens associated with paralysis, immunosuppression, neurological signs, and T-cell lymphomas. MD is controlled in poultry production via live attenuated vaccines. The purpose of the current study was to compare methods for precipitating exosomes from vaccinated and protected chicken sera (VEX) and tumor-bearing chicken sera (TEX) for biomarker analysis of vaccine-induced protection and MD lymphomas respectively. A standard polyethylene glycol (PEG, 8%) method was compared to a commercial reagent (total exosome isolation reagent, TEI) for exosome yield and RNA content. Although exosomes purified by PEG or TEI were comparable in size and morphology, TEI-reagent yielded 3-4-fold greater concentration. Relative expression of 8 out of 10 G. gallus- and MDV1-encoded miRNAs examined displayed significant difference depending upon the precipitation method used. Standard PEG yields comparable, albeit lower amounts of exosomes than the TEI-reagent and a distinctive miRNA composition., Immunology; Virology; Animal science; Transcriptomics; Veterinary medicine; Hematological system; miRNA; Marek's disease virus; Extracellular vesicle; Exosome precipitation.

Despite approval of immunotherapy for a wide range of cancers, the majority of patients fail to respond to immunotherapy or relapse following initial response which may be attributed to immunosuppression co-opted by tumor cells. However, it is challenging to utilize conventional methods to systematically evaluate the potential of tumor intrinsic factors to act as immune regulators in cancer patients. In this study, we designed an unbiased integrative strategy to leverage the complementary strength of in vitro functional genomic screens and multi-omics clinical data to assess the role of individual tumor-intrinsic factors in regulating T cell tumor infiltration and T cell-mediated tumor killing, the two most important rate-limiting steps of cancer immunotherapy. Initially, a genome-wide CRISPR-Cas9 screening system using paired murine tumors and tumor-reactive T cells was employed to globally screen tumor intrinsic factors modulating the tumor sensitivity to T cell-mediated killing. Then, findings from the screening were integrated with the bioinformatics analysis of clinical datasets to further evaluate the role of each tumor intrinsic factor in governing antitumor immunity. The integrative analysis not only successfully identified several novel tumor intrinsic factors as effectors of immune resistance, but also demonstrated the distinct roles of these factors in controlling immune cell trafficking and tumor sensitivity to T cell-mediated killing. Among these factors, candidates controlling both rate-limiting steps were termed as "Dual immune resistance regulators" and the remaining factors whose expression were not associated with tumor immune infiltration were termed as "Cytotoxicity resistance regulators". By selecting PRMT1 and RIPK1 as representatives of the two groups respectively, we confirmed that genetically depletion of PRMT1 and RIPK1 sensitized tumors to T-cell mediated killing via two independent experimental approaches. Furthermore, inhibiting Prmt1 or Ripk1 tumors sensitizes tumors to cancer immunotherapy, such as anti-PD-1/anti-OX40 treatments (Tumor size (mm2) on day 21 after tumor inoculation: for anti-PD-1 treatment, Ctrl 84.05±23.10, PRMT1 KO 7.30±7.81, RIPK1 KO 2.03±4.96; similar results were also observed from anti-OX40 treatment) and extended the survival of tumor-bearing mice. Moreover, by using a RIPK1-specific inhibitor, GSK2982772, we demonstrated that targeting cytotoxicity resistance regulators could also enhance the antitumor activity of T cell-based cancer immunotherapy, despite limited impact on T cell tumor infiltration. Collectively, our data not only demonstrate the distinct immunoregulatory roles and therapeutic potentials of PRMT1 and RIPK1 in T cell-mediated antitumor activity, but also provides a rich resource of novel targets for rational immuno-oncology combinations. Citation Format: Jiakai Hou, Yunfei Wang, Leilei Shi, Yuan Chen, Chunyu Xu, Arash Saeedi, Ke Pan, Ritu Bohat, Nicholas A. Egan, Jodi A. McKenzie, Rina M. Mbofung, Leila J. Williams, Zhenghuang Yang, Ming Sun, Xiaofang Liang, Jordi Rodon Ahnert, Navin Varadarajan, Cassian Yee, Yiwen Chen, Patrick Hwu, Weiyi Peng. The landscape of tumor intrinsic immune regulators revealed by genome-wide CRISPR immune screen integrated with comprehensive clinical data analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1575.

Heterogeneity in host susceptibility is a key determinant of infectious disease dynamics but is rarely accounted for in assessment of disease control measures. Understanding how susceptibility is distributed in populations, and how control measures change this distribution, is integral to predicting the course of epidemics with and without interventions. Using multiple experimental and modeling approaches, we show that rainbow trout have relatively homogeneous susceptibility to infection with infectious hematopoietic necrosis virus and that vaccination increases heterogeneity in susceptibility in a nearly all-or-nothing fashion. In a simple transmission model with an R0 of 2, the highly heterogeneous vaccine protection would cause a 35 percentage-point reduction in outbreak size over an intervention inducing homogenous protection at the same mean level. More broadly, these findings provide validation of methodology that can help to reduce biases in predictions of vaccine impact in natural settings and provide insight into how vaccination shapes population susceptibility., IMPORTANCE Differences among individuals influence transmission and spread of infectious diseases as well as the effectiveness of control measures. Control measures, such as vaccines, may provide leaky protection, protecting all hosts to an identical degree, or all-or-nothing protection, protecting some hosts completely while leaving others completely unprotected. This distinction can have a dramatic influence on disease dynamics, yet this distribution of protection is frequently unaccounted for in epidemiological models and estimates of vaccine efficacy. Here, we apply new methodology to experimentally examine host heterogeneity in susceptibility and mode of vaccine action as distinct components influencing disease outcome. Through multiple experiments and new modeling approaches, we show that the distribution of vaccine effects can be robustly estimated. These results offer new experimental and inferential methodology that can improve predictions of vaccine effectiveness and have broad applicability to human, wildlife, and ecosystem health.

Actin-based motility is central to the pathogenicity of the intracellular bacterial pathogen Shigella . Two Shigella outer membrane proteins, IcsA and IcsP, are required for efficient actin-based motility in the host cell cytoplasm, and the genes encoding both proteins are carried on the large virulence plasmid. IcsA triggers actin polymerization on the surface of the bacterium, leading to the formation of an actin tail that allows both intra- and intercellular spread. IcsP, an outer membrane protease, modulates the amount and distribution of the IcsA protein on the bacterial surface through proteolytic cleavage of IcsA. Transcription of icsP is increased in the presence of VirB, a DNA-binding protein that positively regulates many genes carried on the large virulence plasmid. In Shigella dysenteriae , the small regulatory RNA RyhB, which is a member of the iron-responsive Fur regulon, suppresses several virulence-associated phenotypes by downregulating levels of virB in response to iron limitation. Here we show that the Fur/RyhB regulatory pathway downregulates IcsP levels in response to low iron concentrations in Shigella flexneri and that this occurs at the level of transcription through the RyhB-dependent regulation of VirB. These observations demonstrate that in Shigella species the Fur/RyhB regulatory pathway provides a mechanism to finely tune the expression of icsP in response to the low concentrations of free iron predicted to be encountered within colonic epithelial cells.

Infection of the human host by Shigella species requires the coordinated production of specific Shigella virulence factors, a process mediated largely by the VirF/VirB regulatory cascade. VirF promotes the transcription of virB, a gene encoding the transcriptional activator of several virulence-associated genes. This study reveals that transcription of virB is also regulated by the small RNA RyhB, and importantly, that this regulation is not achieved indirectly via modulation of VirF activity. These data are the first to demonstrate that the regulation of virB transcription can be uncoupled from the master regulator VirF. It is also established that efficient RyhB-dependent regulation of transcription is facilitated by specific nucleic acid sequences within virB. This study not only reveals RyhB-dependent regulation of virB transcription as a novel point of control in the central regulatory circuit modulating Shigella virulence, but also highlights the versatility of RyhB in controlling bacterial gene expression.