Reshaping the Tumor Microenvironment of KRASG12D Pancreatic Ductal Adenocarcinoma with combined SOS1 and MEK Inhibition for Improved Immunotherapy Response.

KRAS inhibitors have demonstrated exciting pre-clinical and clinical responses, although resistance occurs rapidly. Here, we investigate the effects of KRAS-targeting therapies on the tumor microenvironment using a library of KRASG12D, p53 mutant, murine PDAC-derived cell lines (KPCY) to leverage immune-oncology combination strategies for long-term tumor efficacy. Our findings show that SOS1 and MEK inhibitors (SOS1i+MEKi) suppressed tumor growth in syngeneic models and increased intra-tumoral CD8+ T cells without durable responses. scRNA-sequencing revealed an increase in inflammatory cancer associated fibroblasts (iCAFs), M2 macrophages, and a decreased dendritic cell quality that ultimately resulted in a highly immunosuppressive microenvironment driven by IL6+ iCAFs. Agonist CD40 treatment was effective to revert macrophage polarization and overcome the lack of mature antigen presenting DCs after SOS1i+MEKi therapy. Treatment increased the overall survival of KPCY tumor-bearing mice. The addition of checkpoint blockade to SOS1i+MEKi combination resulted in tumor free mice with established immune memory. Our data suggests that KRAS inhibition affects myeloid cell maturation and highlights the need for combining KRAS cancer-targeted therapy with myeloid activation to enhance and prolong anti-tumor effects.

Brief report: STING expressed in tumor and non-tumor compartments has distinct roles in regulating anti-tumor immunity.

Type I interferon-mediated activation of immune cells can facilitate the generation of productive tumor antigen-specific T cell responses in solid tumors. The cGAS/STING DNA sensing pathway is a critical upstream mediator of type I interferon production and is an important regulator of anti-tumor immunity. Numerous STING pathway agonists are now being tested in clinical trials, but the effectiveness of this approach is not yet clear and a better understanding of the relative importance of this pathway in various tumor settings is needed. We have evaluated syngeneic tumor models with different baseline inflammatory states to determine the contributions of STING activity in both tumor and non-tumor cellular compartments to anti-tumor immune responses. We find that productive anti-tumor immune responses in the poorly immunogenic B16F10 model show a strong dependence on STING expression in non-tumor cells. In the immunogenic MC38 model, constitutive STING activation in tumor cells can partially bypass the requirement for STING-dependent activity from immune cells. Our findings reveal multiple, context-dependent roles for STING activity in the regulation of anti-tumor immunity and the response to immunotherapy. In preclinical models where STING is basally active, checkpoint inhibition is more likely to have a therapeutic effect and removal of STING signaling from either the tumor or the non-tumor compartment has a minimal effect. Removal of STING signaling in both, however, diminishes the efficacy derived from checkpoint therapy. Further work is needed to understand the heterogeneity of STING signaling in patients, both in tumor cells and the tumor microenvironment, and the best means of harnessing this pathway to generate anti-tumor immunity and improve therapeutic outcomes.

Immunotherapy combinations overcome resistance to bispecific T cell engager treatment in T cell-cold solid tumors.

Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3ε chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8+ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4+ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell-infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8+ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule-mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.

Spontaneous Differentiation of T Follicular Helper Cells in LATY136F Mutant Mice.

Mice with a mutation at the LAT-PLCγ1 binding site (Y136) have a defect in thymocyte development due to dampened TCR signaling. CD4+ T cells that do reach the periphery are hyper-activated and skewed to Th2. Over time, these mice develop an autoimmune-like syndrome, characterize by overproduction of Th2 cytokines, T cell infiltration into various organs, and B cell activation, isotype switching, and autoantibody production. In this study, we examined IL4 production by CD4+ T cells in the LATY136F mice using the KN2 reporter mice, in which human CD2 expression marks T cells that are actively producing IL4 protein. We showed that these mice had spontaneous Tfh differentiation. Despite the fact that the majority of CD4+ T cells were skewed to Th2 and were GATA3+, only a small subset of them were actively secreting IL4. These T cells were Tfh cells that expressed BCL6 and were localized to B cell-rich germinal centers within the spleen. Interestingly, these Tfh cells expressed high levels of both BCL6 and GATA3. By using LAT conditional knockout mice that inducibly express only the LATY136F allele, we further showed that Tfh cell differentiation was likely the result of defective LAT-PLCγ1 signaling in the periphery. In addition, B cells were required for spontaneous development of Tfh cells and uncontrolled T cell expansion in these mice. Together, these results indicated a novel role for tonic LAT-PLCγ1 signaling in modulating Tfh cell differentiation during development of autoimmune syndrome.

Activity of tumor-associated macrophage depletion by CSF1R blockade is highly dependent on the tumor model and timing of treatment.

Tumor-associated macrophages (TAMs) are abundant in solid tumors where they exhibit immunosuppressive and pro-tumorigenic functions. Inhibition of TAM proliferation and survival through CSF1R blockade has been widely explored as a cancer immunotherapy. To further define mechanisms regulating CSF1R-targeted therapies, we systematically evaluated the effect of anti-CSF1R treatment on tumor growth and tumor microenvironment (TME) inflammation across multiple murine models. Despite substantial macrophage depletion, anti-CSF1R had minimal effects on the anti-tumor immune response in mice bearing established tumors. In contrast, anti-CSF1R treatment concurrent with tumor implantation resulted in more robust tumor growth inhibition and evidence of enhanced anti-tumor immunity. Our findings suggest only minor contributions of CSF1R-dependent TAMs to the inflammatory state of the TME in established tumors, that immune landscape heterogeneity across different tumor models can influence anti-CSF1R activity, and that alternative treatment schedules and/or TAM depletion strategies may be needed to maximize the clinical benefit of this approach.

Single-Cell Analyses Inform Mechanisms of Myeloid-Targeted Therapies in Colon Cancer.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.

Phospholipase D in TCR-Mediated Signaling and T Cell Activation.

Phospholipase D (PLD) is an enzyme that catalyzes the hydrolysis of phosphatidylcholine, the major phospholipid in the plasma membrane, to generate an important signaling lipid, phosphatidic acid. Phosphatidic acid is a second messenger that regulates vesicular trafficking, cytoskeletal reorganization, and cell signaling in immune cells and other cell types. Published studies, using pharmacological inhibitors or protein overexpression, indicate that PLD plays a positive role in TCR-mediated signaling and cell activation. In this study, we used mice deficient in PLD1, PLD2, or both to assess the function of these enzymes in T cells. Our data showed that PLD1 deficiency impaired TCR-mediated signaling, T cell expansion, and effector function during immune responses against Listeria monocytogenes; however, PLD2 deficiency had a minimal impact on T cells. Biochemical analysis indicated that PLD1 deficiency affected Akt and PKCθ activation. In addition, it impaired TCR downregulation and the secondary T cell response. Together, our results suggested that PLD1 plays an important role in T cell activation.

End-of-life planning with frail patients attending general practice: an exploratory prospective cross-sectional study.

BACKGROUND: End-of-life planning means decision making with patients, formulating and recording decisions regarding their end-of-life care. Although clearly linked with benefits including improved quality of life, reduced hospital admissions, and less aggressive medical care, it is still infrequently undertaken and is regarded as challenging by healthcare professionals. AIM: To ascertain the feasibility of improving the identification of patients at high risk of dying in general practice and the acceptability of providing patients identified with an end-of-life planning tool. DESIGN AND SETTING: Exploratory prospective cross-sectional study in four general practices. METHOD: Patients at high risk of dying were identified during routine consulting by their GP, using the Supportive and Palliative Care Indicators Tool (SPICT). Patients identified were invited to participate, and provided with Think Ahead - an end-of-life planning tool, which has been used previously in general practice. Participants completed telephone surveys, assessing their response to Think Ahead, and the acceptability of the GP raising end-of-life issues during routine consulting. RESULTS: Provision of Think Ahead to a purposive sample of preterminal patients identified by GPs was feasible, acceptable to most patients, and somewhat effective in increasing discussion among families and in practice on end-of-life planning. CONCLUSION: The SPICT and Think Ahead tools were mostly acceptable, effective, and enabling of discussions on end-of-life care in general practice.

Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function.

Phospholipase D (PLD) proteins are enzymes that catalyze the hydrolysis of phosphatidylcholine to generate an important signaling lipid, phosphatidic acid. Phosphatidic acid is a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Previous studies using inhibitors and overexpression of PLD proteins indicate that PLD1 and PLD2 play positive roles in FcεRI-mediated signaling and mast cell function. We used mice deficient in PLD1, PLD2, or both to study the function of these enzymes in mast cells. In contrast to published studies, we found that PLD1 deficiency impaired FcεRI-mediated mast cell degranulation; however, PLD2 deficiency enhanced it. Biochemical analysis showed that PLD deficiency affected activation of the PI3K pathway and RhoA. Furthermore, our data indicated that, although PLD1 deficiency impaired F-actin disassembly, PLD2 deficiency enhanced microtubule formation. Together, our results suggested that PLD1 and PLD2, two proteins that catalyze the same enzymatic reaction, regulate different steps in mast cell degranulation.

The Importance of IL-6 in the Development of LAT-Mediated Autoimmunity.

Linker for activation of T cells (LAT) is a transmembrane adaptor protein that is highly tyrosine phosphorylated upon engagement of the TCR. Phosphorylated LAT binds Grb2, Gads, and phospholipase C (PLC)γ1 to mediate T cell activation, proliferation, and cytokine production. T cells from mice harboring a mutation at the PLCγ1 binding site of LAT (Y136F) have impaired calcium flux and Erk activation. Interestingly, these T cells are highly activated, resulting in the development of a lymphoproliferative syndrome in these mice. CD4(+) T cells in LATY136F mice are Th2 skewed, producing large amounts of IL-4. In this study, we showed that the LATY136F T cells could also overproduce IL-6 due to activated NF-κB, AKT, and p38 pathways. By crossing LATY136F mice with IL-6-deficient mice, we demonstrated that IL-6 is required for uncontrolled T cell expansion during the early stage of disease development. Reduced CD4(+) T cell expansion was not due to a further block in thymocyte development or an increase in the number of regulatory T cells, but was caused by reduction in cell survival. In aged IL-6(-/-) LATY136F mice, CD4(+) T cells began to hyperproliferate and induced splenomegaly; however, isotype switching and autoantibody production were diminished. Our data indicated that the LAT-PLCγ1 interaction is important for controlling IL-6 production by T cells and demonstrated a critical role of IL-6 in the development of this lymphoproliferative syndrome.

Impact of biopsychosocial factors on chronic pain in persons with myotonic and facioscapulohumeral muscular dystrophy.

To assess the role of biopsychosocial factors in patients with type 1 myotonic and facioscapulohumeral muscular dystrophy (MMD1/FSHD) with chronic pain. Associations between psychosocial factors were found to be important in other samples of persons with pain and both psychological functioning and pain interference in a sample of patients suffering from MMD/FSHD. Prospective, multiple group, survey study of 182 patients with confirmed MMD1 and FSHD. Participants completed surveys assessing pain interference and psychological functioning, as well as psychosocial, demographic, and injury-related variables. Analyses indicated that greater catastrophizing was associated with increased pain interference and poorer psychological functioning, pain attitudes were significantly related to both pain interference and psychological functioning, and coping responses were significantly related only to pain interference. In addition, greater perceived social support was associated with better psychological functioning. The results support the use of studying pain in persons with MMD/FSHD from a biopsychosocial perspective, and the importance of identifying psychosocial factors that may play a role in the adjustment to and response to pain secondary to MMD/FSHD.