Analyses and Correlation of Pathologic and Ocular Cutaneous Changes in Murine Graft versus Host Disease

Graft versus host disease (GVHD) is initiated by donor allo-reactive T cells activated against recipient antigens. Chronic GVHD (cGVHD) is characterized by immune responses that may resemble autoimmune features present in the scleroderma and Sjogren’s syndrome. Unfortunately, ocular involvement occurs in approximately 60–90% of patients with cGVHD following allo-hematopoietic stem cell transplants (aHSCT). Ocular GVHD (oGVHD) may affect vision due to ocular adnexa damage leading to dry eye and keratopathy. Several other compartments including the skin are major targets of GVHD effector pathways. Using mouse aHSCT models, the objective was to characterize cGVHD associated alterations in the eye and skin to assess for correlations between these two organs. The examination of multiple models of MHC-matched and MHC-mismatched aHSCT identified a correlation between ocular and cutaneous involvement accompanying cGVHD. Studies detected a “positive” correlation, i.e., when cGVHD-induced ocular alterations were observed, cutaneous compartment alterations were also observed. When no or minimal ocular signs were detected, no or minimal skin changes were observed. In total, these findings suggest underlying cGVHD-inducing pathological immune mechanisms may be shared between the eye and skin. Based on the present observations, we posit that when skin involvement is present in aHSCT patients with cGVHD, the evaluation of the ocular surface by an ophthalmologist could potentially be of value.

The versatile regulation of K2P channels by polyanionic lipids of the phosphoinositide and fatty acid metabolism

Work over the past three decades has greatly advanced our understanding of the regulation of Kir K+ channels by polyanionic lipids of the phosphoinositide (e.g., PIP2) and fatty acid metabolism (e.g., oleoyl-CoA). However, comparatively little is known regarding the regulation of the K2P channel family by phosphoinositides and by long-chain fatty acid–CoA esters, such as oleoyl-CoA. We screened 12 mammalian K2P channels and report effects of polyanionic lipids on all tested channels. We observed activation of members of the TREK, TALK, and THIK subfamilies, with the strongest activation by PIP2 for TRAAK and the strongest activation by oleoyl-CoA for TALK-2. By contrast, we observed inhibition for members of the TASK and TRESK subfamilies. Our results reveal that TASK-2 channels have both activatory and inhibitory PIP2 sites with different affinities. Finally, we provided evidence that PIP2 inhibition of TASK-1 and TASK-3 channels is mediated by closure of the recently identified lower X-gate as critical mutations within the gate (i.e., L244A, R245A) prevent PIP2-induced inhibition. Our findings establish that K+ channels of the K2P family are highly sensitive to polyanionic lipids, extending our knowledge of the mechanisms of lipid regulation and implicating the metabolism of these lipids as possible effector pathways to regulate K2P channel activity.

MYC regulates a pan-cancer network of co-expressed oncogenic splicing factors

MYC is dysregulated in >50% of cancers, but direct targeting of MYC has been clinically unsuccessful. Targeting downstream MYC effector pathways represents an attractive alternative. MYC regulates alternative mRNA splicing, a hallmark of cancer, but the mechanistic links between MYC and the splicing machinery remain underexplored. Here, we identify a network of splicing factors (SFs) co-expressed as SF-modules in MYC-active breast tumors. Of these, one is a pan-cancer SF-module, correlating with MYC-activity across 33 tumor types. In mammary cell models, MYC activation leads to co-upregulation of pan-cancer module SFs and to changes in >4,000 splicing events. In breast cancer organoids, co-overexpression of the pan-cancer SF-module is sufficient to induce splicing events that are also MYC-regulated in patient tumors and to increase organoid size and invasiveness, while its knockdown decreases organoid size. Finally, we uncover a pan-cancer splicing signature of MYC activity which correlates with survival in multiple tumor types. Our findings provide insight into the mechanisms and function of MYC-regulated splicing and for the development of therapeutics for MYC-driven tumors.

AML Cell Vaccines Co-Expressing CD80 and IL-15/IL-15 Receptor Alpha Induce Activation and Cytolytic Activity in Post Remission Autologous Patient PBMC Ex Vivo

There is a critical need for more effective therapy for acute myelogenous leukemia (AML). Although many patients achieve remission, most relapse with poor outcomes. Even after allogeneic Stem Cell Transplantation (SCT), 30-50% of patients relapse due to the persistence of residual disease. To address the poor immunogenicity of AML cells and the diminished immune responsiveness of patients, our candidate autologous AML vaccine is lentivirally engineered, in each patient's leukemic cells, to express CD80, IL-15, and IL-15 Receptor alpha (IL-15Rα). In prior studies in a syngeneic 32Dp210 murine AML model, CD80-mediated co-stimulation of T-cells combined with immune activation by the IL-15/IL-15Rα heterodimer showed unprecedented synergy in induction of anti-leukemic cytolytic activity (Shi, Y. et al, 2018). This was observed in both ex vivo co-culture and in vivo where vaccinated leukemic mice had >80% cure rates. No local skin, organ, or systemic toxicity was observed, nor was there evidence of systemic cytokine release of IL-6 or TNFα after SC or IV injection of up to 10 8 transduced irradiated AML cells. We confirmed the feasibility of producing patient-derived AML vaccines by transduction of 16 independent AML samples with a tri-cistronic lentiviral vector (TLV) that contains human CD80, IL-15 and IL-15Rα. Transduction levels were 11-71% of cells (median 38.6%). To define the minimum transduction level required for PBMC activation and to assess synergy of co-expressed human CD80, IL-15, and IL-15Rα, allogenic U937 leukemia cells were initially used as stimulators. Transduced U937 (U937-TLV) had high-level surface expression of CD80 and IL-15, secreted IL-15 (7 ng/ml/24 hours from 2 x 10 6 cells/ml) and activated CD3+ T-cells from an AML patient (Fig.1). Mixtures of irradiated U937-TLV with non-transduced U937 were created at fixed ratios (100%, 80%, 40%, 20%, 10%, 5%, 0%) for overnight co-culture with patient PBMC. At 24 hours, the T-cells were analyzed for activation by measurement of the frequency of CD69+ CD4 or CD8 T cells (Fig. 1), normalized to expression of unstimulated PBMC (0%) and the percentage of maximal CD69 expression with 100% U937-TLV (100%). Background levels of activation due to the presence of allogenic U937 were negligible. Co-culture with as little as 10% transduced U937-TLV reliably activated patient T-cells. To assess the synergy of CD80, IL-15 and IL-15Rα expression, parallel experiments were performed with PBMC co-cultured in IL-15 containing supernatants from U937-TLV cells (Fig. 1). The frequencies of activated T-cells were significantly higher after co-culture with U937/U937-TLV cells than after stimulation with IL-15-containing supernatants from similar ratios of U937/U937-TLV, confirming the synergy of CD80 and IL-15/IL-15Rα in the transduced cells. To better, model the clinical setting, we assessed induction of immune responses of patient T cells to autologous transduced AML. PBMC were stimulated with transduced or non-transduced autologous AML cells vs stimulation with allogeneic U937-TLV, or with anti-CD3/CD28 beads to define maximal stimulation. Negative controls included culture of PBMC alone. All patients had T-cell activation, as measured by induction of CD69, HLA-DR and CD95 (Fas) expression, although there was heterogeneity in the nature of responses, e.g., disparate induction of the markers in individual patients (Fig. 2A and B). Induction of cytotoxic effector pathways was confirmed by detection of CD178 (FasL) and perforin expression (Figure 2C and D). Overall, all patients' PBMC had the capacity to mount T-cell responses of similar magnitude to both allogeneic U937-TLV and autologous vaccine. These studies establish that autologous AML cells transduced with CD80, IL-15 and IL-15Rα can elicit specific anti-leukemic T-cell responses, even in the face of prior lymphodepleting chemotherapy. A strength of this autologous vaccine strategy is that it is agnostic to which AML proteins are immunogenic for each patient. Although uniformly detected, there was heterogeneity in the induction of activation markers and effector pathways, which may reflect host and/or disease-related differences. The mechanisms underlying differences in the nature of responses in patients will be important to understand and will provide the basis for future immune correlative studies for our Phase 1 vaccine trial in transplant ineligible AML patients. Figure 1 Figure 1. Disclosures Kohn: Lyrik Therapeutics: Membership on an entity's Board of Directors or advisory committees; MyoGene Bio: Membership on an entity's Board of Directors or advisory committees; ImmunoVec: Membership on an entity's Board of Directors or advisory committees; Pluto Immunotherapeutics: Membership on an entity's Board of Directors or advisory committees; Allogene: Membership on an entity's Board of Directors or advisory committees; UC Regents: Patents & Royalties; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Sangamo Biosciences: Membership on an entity's Board of Directors or advisory committees.

S1P/S1P2 Signaling Axis Regulates Both NLRP3 Upregulation and NLRP3 Inflammasome Activation in Macrophages Primed with Lipopolysaccharide

The activation of NLRP3 inflammasome is a key factor for various inflammatory diseases. Here, we provide experimental evidence supporting the regulatory role of sphingosine-1-phosphate (S1P) in NLRP3 inflammasome activation in mouse bone-marrow-derived macrophages (BMDMs), along with the S1P receptor subtype involved and underlying regulatory mechanisms. During the priming stage, S1P induced NLRP3 upregulation in BMDMs only when primed with lipopolysaccharide (LPS). In this event, S1P2, but not S1P1, was involved based on the attenuated NLRP3 upregulation with JTE013 (S1P2 antagonist) or S1P2 knockdown. During the activation stage, S1P induced NLRP3 inflammasome activation in LPS-primed BMDMs via caspase-1 activation, interleukin 1β maturation, apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, and IL-1β secretion. Such NLRP3 inflammasome activation was blocked by either pharmacological inhibition or genetic knockdown of S1P2. NF-κB, PI3K/Akt, and ERK1/2 were identified as effector pathways underlying S1P/S1P2 signaling in the regulation of NLRP3 upregulation in LPS-primed BMDMs. Further, reactive oxygen species (ROS) production was dependent on the S1P/S1P2 signaling axis in these cells, and the ROS generated regulate NLRP3 inflammasome activation, but not NLRP3 priming. Collectively, our findings suggest that S1P promotes NLRP3 upregulation and NLRP3 inflammasome activation in LPS-primed BMDMs via S1P2 and subsequent effector pathways.

Escaping KRAS: Gaining Autonomy and Resistance to KRAS Inhibition in KRAS Mutant Cancers

Activating mutations in KRAS are present in 25% of human cancers. When mutated, the KRAS protein becomes constitutively active, stimulating various effector pathways and leading to the deregulation of key cellular processes, including the suppression of apoptosis and enhancement of proliferation. Furthermore, mutant KRAS also promotes metabolic deregulation and alterations in the tumor microenvironment. However, some KRAS mutant cancer cells become independent of KRAS for their survival by activating diverse bypass networks that maintain essential survival signaling originally governed by mutant KRAS. The proposed inducers of KRAS independency are the activation of YAP1 and/or RSK-mTOR pathways and co-mutations in SKT11 (LKB1), KEAP1, and NFE2L2 (NRF2) genes. Metabolic reprogramming, such as increased glutaminolysis, is also associated with KRAS autonomy. The presence or absence of KRAS dependency is related to the heterogeneity of KRAS mutant cancers. Epithelial-to-mesenchymal transition (EMT) in tumor cells is also a characteristic phenotype of KRAS independency. Translationally, this loss of dependence is a cause of primary and acquired resistance to mutant KRAS-specific inhibitors. While KRAS-dependent tumors can be treated with mutant KRAS inhibitor monotherapy, for KRAS-independent tumors, we need an improved understanding of activated bypass signaling pathways towards leveraging vulnerabilities, and advancing therapeutic options for this patient subset.

Infection-induced miR-126 suppresses tsc1- and cxcl12a-dependent permissive macrophages during mycobacterial infection

Regulation of host microRNA (miRNA) expression is a contested node that controls the host immune response to mycobacterial infection. The host must overcome concerted subversive efforts of pathogenic mycobacteria to launch and maintain a protective immune response. Here we examine the role of miR-126 in the zebrafish model of Mycobacterium marinum infection and identify a protective role for this infection-induced miRNA through multiple effector pathways. Specifically, we analyse the impact of the miR-126 knockdown-induced tsc1a and cxcl12a/ccl2/ccr2 signalling axes during early host-M. marinum interactions. We find a strong detrimental effect of tsc1a upregulation that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death despite dramatically higher recruitment of macrophages to the site of infection. We demonstrate that infection-induced miR-126 suppresses tsc1 and cxcl12a expression thus improving macrophage function early in infection, partially through activation of mTOR signalling and strongly through preventing the recruitment of Ccr2+ permissive macrophages, resulting in the recruitment of protective tnfa-expressing macrophages. Together our results demonstrate an important role for infection-induced miR-126 in shaping an effective immune response to M. marinum infection in zebrafish embryos.

Interconnections between Inflammageing and Immunosenescence During Ageing

Acute inflammation is a physiological response to injury or infection, with a cascade of steps that ultimately leads to recruitment of immune cells to clear invading pathogens and heal wounds. However, chronic inflammation arising from continued presence of the initial trigger, or dysfunction of signalling and/or effector pathways, is harmful to health. While successful ageing in older adults including centenarians is associated with low levels of inflammation, elevated inflammation increases the risk of poor health and death [1–3]. Hence inflammation has been described as one of seven pillars of ageing. Age-associated sterile, chronic, and low-grade inflammation is commonly termed inflammageing - it is not simply a consequence of increasing chronological age, but is also a marker of biological ageing, multimorbidity and mortality risk. While inflammageing was initially thought to be caused by “continuous antigenic load and stress”, reports from the last two decades describe a much more complex phenomenon also involving cellular senescence and ageing of the immune system. In this review, we explore the sources and consequences of inflammageing and highlight potential interventions. In particular, we assess the contribution of cellular senescence to age-associated inflammation, identify patterns of pro- and anti-inflammatory markers characteristic of inflammageing, describe alterations in the ageing immune system that lead to elevated inflammation, and finally assess the ways that diet, exercise and pharmacological interventions can reduce inflammageing and thus improve later life health.

Targeting PI3K Pathway in Pancreatic Ductal Adenocarcinoma: Rationale and Progress

Pancreatic ductal adenocarcinoma (PDAC) remains among the deadliest solid tumors that remain treatment-refractory and show a dismal prognosis. More than 90% of PDAC tumors harbor mutations in the K-Ras that exert a strong pro-tumorigenic effect by activating several downstream effector pathways, including phosphatidylinositol-3-kinase (PI3K)-Akt. The role of frequently activated PI3K/Akt pathway in promoting PDAC aggressiveness is well established. Therapeutic approaches targeting PI3K and downstream signaling components in different cellular compartments, including tumor, stromal and immune cells, have directly impacted the tumor burden in this cancer type. Our previous work has demonstrated that targeting the PI3K/Akt/mTOR pathway reduced tumor growth and improved survival in the genetic mouse model of PDAC. Here, we discuss the significance of targeting PI3K signaling and the biological impact of PI3K inhibition in modulating the tumor–stromal immune crosstalk within the microenvironment of pancreatic cancer. Furthermore, this review updates on the current challenges involving the therapeutic implications of targeting this pathway in PDAC.

TREM2 impacts brain microglia, oligodendrocytes and endothelial co-expression modules revealing genes and pathways important in Alzheimer s disease

A microglia response to pathogenic signals in diseases such as Alzheimer s disease (AD) has long been recognised, but recent genetic findings have cemented their direct causal contribution to AD and thus the potential to target them or their effector pathways as a possible treatment strategy. TREM2 is a highly penetrant microglia risk gene for AD, which appears central to the coordination of the damage response by microglia in AD. Its absence has a negative impact on Tau and amyloid symptoms and pathologies. Full knowledge of its pathway and relationships with other brain cells in AD has not been fully characterised, but will be essential to fully evaluate the impact of manipulating this pathway for treatment development and to establish the best targets for achieving this. We used whole genome RNA sequencing of hippocampus and cortical brain samples from control, AD, and AD TREM2 risk carriers to identify TREM2-dependent genes driving changes in pathways, processes and cell types in AD. Through highly influential intra and intermodular hub genes and overall changes in the levels of gene expression, TREM2-DAP12 was found to strongly influence a number of other microglia, oligodendrocyte and endothelial genes, notably those involved in complement and Fcγ receptor function, microglia-associated ribosomal genes and oligodendrocyte genes, particularly proteosomal subunits. These strong TREM2 centred co-expression relationships were significantly disrupted in AD cases with a TREM2 risk variant, revealing for the first time genes and pathways directly impacted by TREM2 in the brains of AD patients. Consistent with its function as a lipid sensor, our data supports a role for TREM2 in mediating oligodendrocyte and/or myelin clearance in AD which may be essential not only for preserving healthy tissue homeostasis but may also serve to minimise the persistence of antigenic peptides and lipids which may lead to detrimental pro-inflammatory sequelae. Further work should expand our knowledge of TREM2 on complement and Fcγ receptor function and its impact on oligodendcrotye and myelin integrity and further evaluate the genes and pathways we have identified as possible treatment targets for AD.