Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer characterized by activating KRAS mutations and TP53 alterations. TP53 missense mutations lose their wild-type tumor-suppressor function. Here, we studied whether p53 missense mutations have potential gain-of-function oncogenic roles and their impact on cancer-cell-intrinsic gene expression and the tumor immune microenvironment (TME) in PDAC. p53R172H established an immunosuppressive TME and impaired the efficacy of immune checkpoint inhibitors (ICIs) by regulating a distinct set of chemokines. Among these, tumor-specific reduction of Cxcl1, which encodes a chemoattractant for neutrophils, promoted T cell infiltration and decreased tumor growth. Mechanistically, p53R172H occupied the distal enhancers of Cxcl1 and amplified its expression. These enhancers were responsible for Cxcl1 expression and were essential for its immunosuppressive function. Nuclear factor κB (NF-κB) was a critical cofactor required for p53R172H occupancy at these enhancers. Thus, a common mutation in a tumor-suppressor transcription factor appropriates enhancers, thereby stimulating chemokine expression and establishing an immunosuppressive TME that diminishes ICI efficacy in PDAC.

Tumor cell-intrinsic signaling pathways can drastically affect the tumor immune microenvironment, promoting tumor progression and resistance to immunotherapy by excluding immune cell populations from the tumor. Several tumor cell-intrinsic pathways have been reported to modulate myeloid-cell and T-cell infiltration, creating "cold" tumors. However, clinical evidence suggests that excluding cytotoxic T cells from the tumor core also mediates immune evasion. In this study, we find that tumor cell-intrinsic SOX2 signaling in non-small cell lung cancer induces the exclusion of cytotoxic T cells from the tumor core and promotes resistance to checkpoint blockade therapy. Mechanistically, tumor cell-intrinsic SOX2 expression upregulates CCL2 in tumor cells, resulting in increased recruitment of regulatory T cells (Treg). CD8+ T-cell exclusion depended on Treg-mediated suppression of tumor vasculature. Depleting tumor-infiltrating Tregs via glucocorticoid-induced TNF receptor-related protein restored CD8+ T-cell infiltration and, when combined with checkpoint blockade therapy, reduced tumor growth. These results show that tumor cell-intrinsic SOX2 expression in lung cancer serves as a mechanism of immunotherapy resistance and provide evidence to support future studies investigating whether patients with non-small cell lung cancer with SOX2-dependent CD8+ T-cell exclusion would benefit from the depletion of glucocorticoid-induced TNFR-related protein-positive Tregs.

Abstract Pancreatic ductal Adenocarcinoma (PDAC) is an aggressive malignancy complicated by poor early diagnosis and a lack of response to traditional treatments. It is characterized by a desmoplastic stroma, a lack of infiltration and activation of T cells, and a low mutational burden. The genetic landscape of PDAC is defined by activating KRAS mutations (~90%) and p53 alterations (~70%), but the molecular switches perturbed by these genetic aberrations remain unclear. p53 missense mutations, unlike mutations resulting in the loss of p53, are considered to acquire tumor-supporting functions. But these novel functions remain uncharacterized. The ambiguity in the molecular mechanism of mutant-p53 is further exacerbated by the existence of various types of p53 mutations. The majority of p53 mutations are missense mutations in the DNA binding domain. The repertoire of transcription factors (TFs) it can interact with and the vast regulatory landscape of each TF—composed of gene promoters and distal enhancers—present obstacles in understanding the molecular mechanisms promoting PDAC. In this study, we examined how a common p53 missense mutation in PDAC plays a role in weakening the Immune checkpoint Inhibitors (ICIs) efficacy. Using cells derived from a genetically engineered mouse model (GEMM) of PDAC with activating KRAS mutation (KrasG12D/+) and a p53 missense mutation (p53R172H/-), we found that the PDAC tumorigenesis and resistance to ICIs are dependent on the mutant-p53. We used isogenic p53-null PDAC cells and the restoration of p53R172H in p53-null cells to demonstrate the role of p53R172H in controlling the expression of immunosuppressive chemokine genes such as Cxcl1. p53R172H deletion attenuated PDAC tumor growth, increased the influx of cytotoxic T-cells, and sensitized the tumor to ICIs. The p53R172H-mediated TME reprogramming was replicated by the deletion of the Cxcl1 gene, suggesting the anti-tumorigenic effect of p53R172H was mediated by the Cxcl1 gene. We probed the mechanism of Cxcl1 expression dependence on p53R172H. We found that in conjunction with NF-kB, p53R172H occupies the distal transcription regulatory elements (dTREs) of the Cxcl1 gene harboring NF-kB binding sites. Strikingly, deletion of the Cxcl1 dTREs in PDAC cells recapitulates the phenotypes of p53R172H deletion and Cxcl1 deletion in terms of tumor size, immune landscape of the TME, and ICI responsiveness. Furthermore, we examined the interplay between p53R172H and NF-kB and found that the p53R172H physically interacts with the NF-kB subunit RelA and facilitates its nuclear translocation. Overall, we characterize how a common p53 mutation in PDAC co-opts non-coding regulatory DNA to augment the expression of selective chemokine genes and establishes an immunosuppressive TME to shield the therapeutic benefits of ICIs. Citation Format: Dig B. Mahat, Heena Kumra, Emily Metcalf, Sarah Castro, Kim Nguyen1, Arundeep Singh, William W. Ho, Ivy Chen, Brandon Sullivan, Leon Yim, Enrico Moiso, Vikash Chauhan, Hernandez Moura Silva, Stefani Spranger, Rakesh Jain, Phillip A. Sharp. Mutant-p53 amplifies Cxcl1 expression from distal enhancers blunting immune checkpoint inhibition efficacy in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B087.

Background Myeloid cells, unlike other immune cells such as T cells or NK cells, are known residents in the solid tumor microenvironment (TME). In the absence of checkpoints and in proinflammatory conditions, M1 macrophages are known to be capable of direct phagocytosis of tumor cells and can present tumor-associated antigens to the host immune system. However, the immunosuppressive conditions within the TME restrict and limit the anti-tumor response of tumor associated macrophages (TAMs), including their ability to recruit and activate other immune cells against the tumor. Engineered CAR-Monocytes can serve a unique function in cell therapy by bridging a key gap in the treatment of solid tumors. We are developing an autologous engineered CAR-M cell therapy product targeting Glypican-3 to treat hepatocellular carcinoma. The CAR serves as a homing ‘GPS’ signal for trafficking directly to the tumor site, and directs phagocytosis specifically at targeted tumor cells. Our proprietary M83.CAR molecule contains a macrophage-specific costimulatory domain that significantly increases the phagocytosis function of the CAR-M cells. Furthermore, we have demonstrated that the M83.CAR-M cells are not inhibited by the prevalent CD47 ‘do not eat me’ checkpoint, which is known to restrict myeloid function in the TME. Methods Primary hematopoietic stem cells (HSCs) were harvested and engineered to express a CAR molecule by lentivirus transduction generating CAR-HSCs. The CAR-HSCs underwent our proprietary ex-vivo HSC differentiation process to yield CAR-Monocytes. Results Effective phagocytosis of engineered CAR-M cells is central to subsequent mechanisms of actions that can elicit robust anti-tumor immunity against patient-specific neoantigens. However, the first barrier to overcome is effective infiltration of engineered CAR-M into the tumor from the periphery. We have demonstrated that our engineered CAR-Monocyte drug product can successfully home to the targeted tumor specifically, from the periphery. Subsequent in situ differentiation of CAR-monocytes into CAR-macrophages enables robust tumor cell phagocytosis, the central mechanism that results in the following: 1) proinflammatory shift in the TME, 2) recruitment of APCs and immune cells, 3) activation of T-cells against tumor neoantigens. Conclusions The above summarizes a unique outcome of the CAR-M mechanism of action that is not capitulated by CAR-T or CAR-NK cells. Leveraging and further enhancing CAR-M function could lead to the rejection of the tumor and its metastases, particularly in combination with other immune-modulating therapies. Our proprietary HSC-derived CAR-M platform yields a unique product that demonstrates durability and superior function, which we expect to translate to the clinic.

Local environmental factors influence CD8+ T cell priming in lymph nodes (LNs). Here, we sought to understand how factors unique to the tumor-draining mediastinal LN (mLN) impact CD8+ T cell responses toward lung cancer. Type 1 conventional dendritic cells (DC1s) showed a mLN-specific failure to induce robust cytotoxic T cells responses. Using regulatory T (Treg) cell depletion strategies, we found that Treg cells suppressed DC1s in a spatially coordinated manner within tissue-specific microniches within the mLN. Treg cell suppression required MHC II-dependent contact between DC1s and Treg cells. Elevated levels of IFN-γ drove differentiation Treg cells into Th1-like effector Treg cells in the mLN. In patients with cancer, Treg cell Th1 polarization, but not CD8+/Treg cell ratios, correlated with poor responses to checkpoint blockade immunotherapy. Thus, IFN-γ in the mLN skews Treg cells to be Th1-like effector Treg cells, driving their close interaction with DC1s and subsequent suppression of cytotoxic T cell responses.

Background Checkpoint blockade therapy (CBT) can effectively treat lung tumors; however, only 30% of lung cancer patients respond to CBT [1]. Clinical data showed that patients with low T cell infiltration in the tumor microenvironment (TME) respond poorly to CBT [2]. Gaining insight into the molecular and immunological mechanism that impact the entry of CD8 T cells into the TME will enable the development of innovative therapeutic approaches that can be used in combination to effectively treat individuals with cancer. Using a T cell gene signature, we segregated NSCLC patients into T cell-infiltrated and non-T cell-infiltrated and found that Sox2 upregulation correlates with a lack of T cell infiltration. Here we aimed to investigate how high expression of Sox2 in tumors mediates immune evasion in NSCLC. Methods We overexpressed Sox2 in a mouse lung adenocarcinoma cell line driven by KrasG12D/+ and Tp53-/-. SOX2-positive (KPS2) and control (KPCt) tumor cells were inoculated subcutaneously or via tail vein injection to induce lung metastasis. We treated tumors with anti-PD-L1 and anti-CTLA-4 blocking antibodies and analyzed for tumor burden. T cell infiltration was evaluated by fluorescence microscopy. KPCt and KPS2 cell lines were engineered to express the model antigen SIY to characterize tumor-specific T-cell responses. Regulatory T cells were targeted by systemic depletion using the Foxp3.DTR mouse model or via neutralization of DKK1 using the mDKN-01 antibody. Finally, we assessed the therapeutic benefit of combining anti-DKK1 treatment with CBT to treat SOX2 tumors. Results We found that Sox2 overexpression induces resistance to CBT mediated by CD8 T cell exclusion from the tumor core. Analysis of tumor-reactive T cells indicated that T cell priming and differentiation into cytotoxic effector T cells were unaffected. However, cytotoxic CD8 T cells failed to infiltrate KPS2 tumors while enriched in the peritumoral regions. Interestingly, we found a high density of regulatory T cells in the peritumoral area. Depletion of regulatory T cells significantly improved the activation of the tumor vasculature and the infiltration of cytotoxic CD8 T cells into the tumor core. Furthermore, we showed that combining a neutralizing antibody against DKK1 with CBT significantly reduced the density of regulatory T cells in the TME, increased CD8 T cell infiltration, and improved tumor control. Conclusions Our results show that tumor cell-intrinsic activation of Sox2 in NSCLC promotes immune evasion and contributes to immunotherapy resistance by retaining effector CD8 T cells outside of the tumor mass. Acknowledgements This work was supported by NCI K99/R00 award, the Ludwig Center at MIT, the SITC-Nektar Therapeutics Equity and Inclusion in Cancer Immunotherapy Fellowship, and Leap Therapeutics Inc. References Hellmann, M. D., Paz-Ares, L., Bernabe Caro, R., Zurawski, B., Kim, S. W., Carcereny Costa, E., Park, K., Alexandru, A., Lupinacci, L., de la Mora Jimenez, E., Sakai, H., Albert, I., Vergnenegre, A., Peters, S., Syrigos, K., Barlesi, F., Reck, M., Borghaei, H., Brahmer, J. R., O’Byrne, K. J., Geese, W. J., Bhagavatheeswaran, P., Rabindran, S. K., Kasinathan, R. S., Nathan, F. E. & Ramalingam, S. S. Nivolumab plus Ipilimumab in Advanced Non-Small-Cell Lung Cancer. N Engl J Med 2019;381, 2020−2031, doi:10.1056/NEJMoa1910231. Chen, D. S. & Mellman, I. Elements of cancer immunity and the cancer-immune set point. Nature 2017;541, 321−330, doi:10.1038/nature21349. Ethics Approval All mouse experiments were approved by MIT’s Committee on Animal Care (CAC) – PHS Animal Welfare Assurance # D16–00078 (A3125–01).

Background Although failure to respond to checkpoint blockade immunotherapies is frequently associated with a lack of T-cell infiltration into the tumor, clinical data suggests that in patients with lung cancer, T-cell-inflamed tumors can also be resistant to therapy.1 Work by us identified that checkpoint blockade immunotherapy resistance in T-cell-inflamed lung cancer is driven by lung cancer-specific CD8+ T-cell dysfunction, characterized by reduced cytolytic capacity and established during priming in the mediastinal lymph nodes (mLN).2 In this study, we sought to uncover lung-specific mechanisms that blunt priming of anti-tumor cytotoxic T-cell responses. Methods To study T-cell priming against lung cancer, we implanted a syngeneic lung cancer cell line (KP) orthotopically in the lungs or subcutaneously in the flanks of C57BL/6 mice. Immune subsets were profiled using flow cytometry, immunofluorescence staining and RNA-sequencing. Immunological mechanism was dissected using adoptive T-cell transfers, bone marrow chimeras and ex vivo co-cultures. Results Both lung and flank KP tumors resulted in type-1-conventional dendritic cell (DC1)-dependent expansion of tumor-reactive T-cells, however, CD8+ T-cells primed in response to lung tumors in the mLN failed to upregulate key markers of effector CD8+ T-cell differentiation, namely CD25 and Granzyme B. Comparing DC1 from tumor-draining inguinal (iLN) and mLN revealed equivalent antigen load, but reduced expression of CD80, CD86 and IL-12 on mLN-derived DC1. Regulatory T-cell (Treg) depletion rescued both stimulatory molecule expression on DC1 and cytotoxic T-cell priming in the tumor-draining mLN, suggesting that lung CD8+ T-cell dysfunction required the local presence of Treg during priming. Ex vivo co-cultures validated that DC1 and Treg were required and sufficient to induce dysfunctional CD8+ T-cells. This immunosuppression was spatially coordinated within tissue-specific LN microniches and required antigen-specific contact between DC1 and Tregs, as abrogating MHCII-dependent Treg:DC1 interactions restored DC1 capacity to prime cytotoxic T-cell responses against lung tumors. The lung-specific suppression was associated with clonally expanded CXCR3+ TH1-like effector Tregs, which were induced upon interferon sensing in the mLN. Consequently, interferon-gamma neutralization early during tumor induction could prevent the immunosuppression and restore cytotoxic T-cell priming in the mLN. Similarly, in cancer patients, interferon-sensing CXCR3+ Tregs but not CD8+/Treg ratios correlated with resistance to checkpoint blockade immunotherapy. Conclusions Our work suggests that the functional quality of Tregs, specifically the interferon-induced CXCR3+ TH1-like effector state, rather than Treg quantity, is instrumental in restraining tumor-reactive T-cell responses and represents a critical barrier to productive anti-tumor immunity. Acknowledgements This work was supported by the Pew Stewart scholarship, the Koch Institute Frontier Research program, the Ludwig Center at MIT and the MIT School of Science Fellowship in Cancer Research. References Doroshow DB, Sanmamed MF, Hastings K, Politi K, Rimm DL, Chen L, Melero I, Schalper KA, and Herbst RS. Immunotherapy in non-small cell lung cancer: facts and hopes. Clin Cancer Res 2019;25:4592–602. Horton BL, Morgan DM, Momin N, Zagorulya M, Torres-Mejia E, Bhandarkar V, Wittrup KD, Love JC, Spranger S. Lack of CD8(+) T cell effector differentiation during priming mediates checkpoint blockade resistance in non-small cell lung cancer. Sci Immunol. 2021;6:eabi8800. Ethics Approval All mouse experiments in this study were approved by MIT9s Committee on Animal Care (CAC) – DHHS Animal Welfare Assurance £ D16-00078.

Although failure to respond to checkpoint blockade immunotherapies (CBT) is frequently associated with a lack of T cell infiltration into the tumor, emerging clinical data suggests that specifically in patients with lung cancer, T cell-inflamed tumors can also be resistant to therapy.1 Recent work by our group identified that immunotherapy resistance in a T cell-inflamed pre-clinical mouse model of lung cancer is driven by a lung cancer-specific CD8+ T cell dysfunctional program (TLdys), characterized by blunted production of IFNg and reduced cytolytic capacity. Intriguingly, this TLdysprogram is established during priming in the tumor-draining mediastinal lymph nodes (mLN). Understanding the lung-specific mechanisms blunting the activation of anti-tumor T cell responses could enable development of novel therapies needed to improve outcomes of patients with CBT-resistant T cell-inflamed lung cancer.To study anti-tumor immune responses against lung tumors, a syngeneic lung cancer cell line (KP) was implanted orthotopically or subcutaneously into C57BL/6 mice. KP cells were engineered to express SIINFEKL and ZsGreen to enable studies of tumor-reactive T cells and antigen uptake by dendritic cells (DC).Lung KP tumors led to the induction of tumor-reactive TLdys CD8+ T cells lacking CD25 and GzmB in the mLN, in contrast to subcutaneous KP tumors, which induced CD25high GzmBhigh tumor-reactive CD8+ T cells in the inguinal LN (iLN). Mouse models lacking DC1 revealed that DC1 are necessary to prime tumor-reactive CD8+ T cells in both LNs. Flow cytometry characterization of DC1 from LNs revealed equivalent levels of antigen load, but reduced levels of costimulatory molecules CD80, CD86 and the cytokine IL-12 in the mLN compared to iLN, suggesting a blunted stimulatory capacity in the lung setting. Regulatory T cell (Treg) depletion using FoxP3DTR mice rescued expression of effector T cell priming in tumor-draining mLN, suggesting that TLdys induction requires the presence of local Treg. Ex vivo co-cultures of antigen-specific CD8+ T cells with DC1 and Treg sorted from the mLN fully recapitulated the in vivo observation, suggesting that both DC1 and Treg are required and sufficient for TLdys induction. Blockade of the MHCII-dependent DC1:Treg interaction restored an effector-like profile of tumor-reactive CD8+ T cells.Treg restrain DC1 stimulatory function in the tumor-draining mLN, leading to the induction of lung cancer-specific dysfunction in tumor-reactive CD8+ T cells and thus rendering the T cell response refractory to CBT-mediated reinvigoration. Blockade of Treg:DC1 interactions can restore priming of lung cancer-reactive effector T cell responses.Pew-Stewart Scholarship, Training grantHerbst RS, et al. Predictive correlates of response to the anti-PD-L1 antibody MPDL3280A in cancer patients. Nature 2014;515:563–567.All mouse experiments in this study were approved by MIT's Committee on Animal Care (CAC) - DHHS Animal Welfare Assurance # D16-00078

Tumor-infiltrating dendritic cells (DCs) assume varied functional states that impact anti-tumor immunity. To delineate the DC states associated with productive anti-tumor T cell immunity, we compared spontaneously regressing and progressing tumors. Tumor-reactive CD8+ T cell responses in Batf3-/- mice lacking type 1 DCs (DC1s) were lost in progressor tumors but preserved in regressor tumors. Transcriptional profiling of intra-tumoral DCs within regressor tumors revealed an activation state of CD11b+ conventional DCs (DC2s) characterized by expression of interferon (IFN)-stimulated genes (ISGs) (ISG+ DCs). ISG+ DC-activated CD8+ T cells ex vivo comparably to DC1. Unlike cross-presenting DC1, ISG+ DCs acquired and presented intact tumor-derived peptide-major histocompatibility complex class I (MHC class I) complexes. Constitutive type I IFN production by regressor tumors drove the ISG+ DC state, and activation of MHC class I-dressed ISG+ DCs by exogenous IFN-β rescued anti-tumor immunity against progressor tumors in Batf3-/- mice. The ISG+ DC gene signature is detectable in human tumors. Engaging this functional DC state may present an approach for the treatment of human disease.

Background Conventional dendritic cells (cDC) are critical mediators of protective anti-tumor CD8+ T-cell responses.1 Batf3-driven DC1 are the predominant cDC subset driving anti-tumor immunity due to their specialized ability to cross-present antigens for T-cell activation.2–4 However, the contribution of other tumor-infiltrating DC subsets such as CD11b+ DC2 to anti-tumor immunity remains poorly characterized. Recent studies suggest that under inflammation, DC subsets can exist in various functional states with differential impacts on their stimulatory potential.5–7 In this study, we sought to dissect the contributions of distinct DC states during a productive or dysfunctional anti-tumor immune response. A nuanced understanding of DC activation states in tumors and the signals that drive them carries therapeutic potential to modulate anti-tumor immunity and enhance immunotherapy responses. Methods We compared the DC infiltrate of a regressing tumor and a progressing tumor to study DC states. Flow immunophenotyping and RNA-sequencing was performed to profile the intratumoral DC compartment. Sorted DC subsets were co-cultured with T-cells ex vivo to evaluate their stimulatory capacity. Cross-dressing (in vivo/ex vivo) was assayed by staining for transfer of tumor-derived H-2b MHC complexes to MHC-mismatched or β2M-deficient DC. Results Anti-tumor CD8+ T-cell responses in Batf3-/- mice lacking DC1 were maintained in regressor tumors but not progressor tumors, suggesting DC1-independent anti-tumor immunity. Functional assays and RNA-sequencing of the intratumoral DC compartment of regressor tumors revealed a Zbtb46-dependent CD11b+ cDC activation state expressing an interferon-stimulated gene signature (ISG+ DC) that was critical for driving optimal anti-tumor CD8+ T-cell responses. Sorted ISG+ DC could activate CD8+ T-cells similar to DC1. Unlike cross-presenting DC1, however, ISG+ DC acquired antigens by cross-dressing with tumor-derived peptide-MHC, thereby bypassing the requirement for cross-presentation to initiate CD8+ T-cell-immunity. Interestingly, ISG+ DC were enriched in regressor tumors compared to progressor tumors, and this was attributable to constitutive tumor cell-intrinsic type-I-interferon (IFN-I) production in regressor tumors. Ablation of tumor cell-derived IFN-I in regressor tumors led to complete loss of anti-tumor T-cell responses in Batf3-/- mice. Conversely, addition of IFNβ to progressor tumors induced ISG+ DC and rescued anti-tumor T-cell responses in Batf3-/- mice. Conclusions We identified a novel IFN-I-induced activation state of CD11b+ cDC, called ISG+ DC, that was capable of driving anti-tumor CD8+ T cell immunity by cross-dressing with tumor-derived pMHC complexes in the absence of DC1. Engaging additional functional states of DC, such as ISG+ DC, will strengthen anti-tumor immunity and may improve immunotherapy responses. References Merad M, et al. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu Rev Immunol 2013;31:563–604 Hildner K, et al. Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity. Science 2008;322(5904)1097–100. Broz ML, et al. Dissecting the tumor myeloid compartment reveals rare activating antigen-presenting cells critical for T cell immunity. Cancer Cell 2014;26(5):638–52. Roberts EW, et al. Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in Melanoma. Cancer Cell 2016;30(2):324–336. Maier B, et al. A conserved dendritic-cell regulatory program limits antitumour immunity. Nature 2020;580(7802):257–262. Bosteels C, et al. Inflammatory Type 2 cDCs acquire features of cDC1s and macrophages to orchestrate immunity to respiratory virus infection. Immunity 2020;52(6):1039–1056.e9. Zilionis R, et al. Single-cell transcriptomics of human and mouse lung cancers reveals conserved myeloid populations across individuals and species. Immunity 2019;50(5):1317–1334.e10.