Search

The molecular mechanisms of luminal cell differentiation are not understood well enough to determine how differentiation goes awry during oncogenesis. Using RNA-Seq analysis, we discovered that CREB1 plays a central role in maintaining new luminal cell survival and that oncogenesis dramatically changes the CREB1-induced transcriptome. CREB1 is active in luminal cells, but not basal cells. We identified ING4 and its E3 ligase, JFK, as CREB1 transcriptional targets in luminal cells. During luminal cell differentiation, transient induction of ING4 expression is followed by a peak in CREB1 activity, while JFK increases concomitantly with CREB1 activation. Transient expression of ING4 is required for luminal cell induction; however, failure to properly down-regulate ING4 leads to luminal cell death. Consequently, blocking CREB1 increased ING4 expression, suppressed JFK, and led to luminal cell death. Thus, CREB1 is responsible for the suppression of ING4 required for luminal cell survival and maintenance. Oncogenic transformation by suppressing PTEN resulted in constitutive activation of CREB1. However, the tumor cells could no longer fully differentiate into luminal cells, failed to express ING4, and displayed a unique CREB1 transcriptome. Blocking CREB1 in tumorigenic cells suppressed tumor growth in vivo, rescued ING4 expression, and restored luminal cell formation, but ultimately induced luminal cell death. IHC of primary prostate tumors demonstrated a strong correlation between loss of ING4 and loss of PTEN. This is the first study to define a molecular mechanism whereby oncogenic loss of PTEN, leading to aberrant CREB1 activation, suppresses ING4 expression causing disruption of luminal cell differentiation.

While CD8+ cytotoxic T cells are clearly critical for identification and elimination of cancer cells, factors concentrated within the tumor microenvironment drive their altered differentiation to a hypofunctional, short-lived state termed exhaustion. Exhaustion is a progressive lineage, and it is now clear that the most terminally exhausted T (tTexh) cells are not the targets of checkpoint blockade immunotherapy but rather serve as factors that limit immunotherapeutic efficacy. Compared directly, tumor-infiltrating CD8+ tTexh cells bear notable phenotypic similarity to CD4+Foxp3+ regulatory T (Treg) cells, suggesting beyond loss of proinflammatory function, tTexh cells may be directly anti-functional and constrain tumor specific immunity. Here we show, when isolated from the same tumor microenvironment, terminally exhausted CD8+ T cells carried similar capacity to suppress T cell proliferation as CD4+Foxp3+ Treg cells. Unlike Treg cells, tTexh cells suppress solely via the ectonucleotidase CD39, which serves to deplete extracellular ATP (eATP) and produce an adenosinergic environment. CD39 expression in tTexh cells is driven by exposure to tumor hypoxia, such that therapeutic targeting of hypoxia limits tTexh suppression and support responses to immunotherapy. Specific deletion of CD39 in CD8+ T cells slows tumor progression and improves response to checkpoint blockade, highlighting a major role for immunosuppression by exhausted T cells in cancer. Thus, tTexh cells are an unappreciated immunoregulatory population and for full immunotherapeutic efficacy, strategies should be designed to either limit their generation, reprogram their immunosuppressive nature, or remove them from the tumor microenvironment.

Background Regulatory T (Treg) cells are vital for preventing autoimmunity but are a major barrier to robust cancer immunity as the tumor microenvironment (TME) recruits and promotes their function. The deregulated cellular metabolism of tumor cells leads to a metabolite-depleted, hypoxic, and acidic TME. While the TME impairs the effector function of highly glycolytic tumor infiltrating CD8 T cells, Treg cell suppressive function is maintained. Further, studies of in vitro induced and ex vivo Treg cells reveal a distinct metabolic profile compared to effector T cells. Thus, it may be that the altered metabolic landscape of the TME and the increased activity of intratumoral Treg cells are linked. Methods Flow cytometry, isotopic flux analysis, Foxp3 driven Cre-lox, glucose tracers, Seahorse extracellular flux analysis, RNA sequencing. Results Here we show Treg cells display heterogeneity in terms of their glucose metabolism and can engage an alternative metabolic pathway to maintain their high suppressive function and proliferation within the TME and other tissues. Tissue derived Treg cells (both at the steady state and under inflammatory conditions) show broad heterogeneity in their ability to take up glucose. However, glucose uptake correlates with poorer suppressive function and long-term functional stability, and culture of Treg cells in high glucose conditions decreased suppressive function. Treg cells under low glucose conditions upregulate genes associated with the uptake and metabolism of the glycolytic end-product lactic acid. Treg cells withstand high lactate conditions, and lactate treatment prevents the destabilizing effects of high glucose culture. Treg cells utilize lactate within the TCA cycle and generate phosphoenolpyruvate (PEP), a critical intermediate that can fuel intratumoral Treg cell proliferation in vivo. Using mice with a Treg cell-restricted deletion of lactate transporter Slc16a1 (MCT1) we show MCT1 is dispensable for peripheral Treg cell function but required intratumorally, resulting in slowed tumor growth and prolonged survival. Conclusions These data support a model in which Treg cells are metabolically flexible such that they can utilize ‘alternative’ metabolites present in the TME to maintain their suppressive identity. Further, our studies support the notion that tumors avoid immune destruction not only by depriving effector T cells of essential nutrients, but also by metabolically supporting regulatory T cells.

Background Durable clinical responses to immune checkpoint blockade (ICB) occur in a limited fraction of patients. We thus hypothesized that the characteristic tumor metabolic switch towards aerobic glycolysis could contribute to ICB resistance. High glucose consumption and lactate production by tumor cells can indeed restrict nutrient availability for tumor-infiltrating T cells, which also rely on glycolysis to proliferate and function. Therefore, we investigated whether targeting tumor glucose metabolism potentiates ICB anti-tumor activity. Methods We modeled tumor-selective glycolysis inhibition by knocking down the critical glycolytic enzyme lactate dehydrogenase A (LDHA-KD) in the murine metastatic breast carcinoma 4T1 and melanoma B16, which are known immune-refractory tumor models. Anti-CTLA-4 and anti-PD-1 were tested in immunocompetent mice orthotopically implanted with control vs. LDHA-KD tumor cells. Changes in glucose metabolism were assessed by Seahorse and fluorescent-glucose flow-cytometry staining. Changes in immune cells were measured by multiparameter flow cytometry. Glucose-dependent effects of anti-CTLA-4 in regulatory T cells (Tregs) were tested in standard suppression assays with increasing glucose concentration (0.5–10 mM). Pearson correlations between glycolysis and intra-tumor immune-cell infiltration by CIBERSORT immune-deconvolution method were analyzed in bulk RNA-sequencing data sets from human and murine tumors treated with ICB. Results Comparison of ICB activity in LDHA-KD vs. control tumor-bearing mice revealed improved anti-tumor effects and overall survival in the setting of glycolysis-defective tumors specifically upon CTLA-4 blockade. Anti-tumor CD8+ T-cell responses correlated with Treg phenotypic and functional destabilization in anti-CTLA-4-treated LDHA-KD tumors. CTLA-4 blockade led to CTLA-4 and CD25 downregulation associated with increased IFN-gamma and TNF-alpha production in Tregs from glycolysis-defective vs. control tumors. We next mimicked high- vs. low-glycolysis tumor microenvironment (TME) in vitro using control vs. LDHA-KD tumor co-cultures with Tregs, control vs. LDHA-KD tumor-conditioned media or directly modulating glucose concentrations. In these assays, we observed that CTLA-4 blockade promotes IFN-gamma±TNF-alpha production and glucose uptake by Tregs and more efficiently counteracts Treg suppression and enhances CD28 co-stimulation at higher glucose concentrations. Lastly, by interrogating transcriptomic data from human melanoma and murine 4T1 tumors, we found that CTLA-4 blockade promotes immune-cell infiltration and metabolic fitness especially in glycolysis-defective tumors. Conclusions Our findings indicate that increasing glucose availability in the TME may improve anti-CTLA-4 therapeutic activity and reveal a new mechanism through which CTLA-4 blockade interferes with Treg immunosuppression in a glucose-dependent manner. These results suggest that CTLA-4 blockade can be more effective in tumors with low glycolysis and/or can be best exploited in combination with inhibitors of tumor glycolysis.

Background Blockade of co-inhibitory ‘checkpoint’ molecules, PD-1 and CTLA-4, has induced impressive clinical responses in advanced tumors; yet only in a subset of patients.1–3 Limited success with checkpoint blockade therapy suggests other cell extrinsic or intrinsic mechanisms may be dampening an effective immune response. Cytotoxic CD8+ T cells (CTL) encountering chronic antigen and metabolic restriction can differentiate to a terminally exhausted state (Texh), marked by hyporesponsiveness and metabolic, epigenetic, and transcriptional dysfunction.4–8 While enrichment of this population in tumor is a negative prognostic factor,9–10 it remains unclear whether Texh are simply non-functional or instead possess tolerogenic or suppressive properties. Transcriptional profiling of tumor-infiltrating PD-1int (progenitor exhausted) CTL versus PD-1hiTIM-3+ (terminally exhausted; Texh), reveals that exhausted cells express a pattern of genes associated with immune suppression. We hypothesize that Texh potentiate the suppressive microenvironment of solid tumor by autoregulation and inhibition of local immune responses. Methods T cell populations were isolated from murine melanoma–B16-F10 or a lab-generated melanoma clone of the spontaneous BREF/PTEN model–by expression of inhibitory receptors and assayed in tandem in microsuppression assays. Murine melanoma clones with inhibited oxidative metabolism were generated by CRISPR-Cas9 deletion and validated for ablated mitochondrial respiration by extracellular flux analysis. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery. Results When sorted directly from tumor, PD-1hiTim3+ Texh, but not progenitor exhausted PD-1int CTL, induce marked suppression of T cell effector responses, comparable to Foxp3+ Treg from the same environment. Expression of the ectonucleotidase, CD39, is uniquely expressed in Texh and increases as T cells differentiate towards exhaustion. Genetic deletion of CD39 in Texh eliminates the regulatory phenotype of tumor-infiltrating Texh and enforced CD39 expression on effector T cells can inhibit T cell receptor signaling and downstream function. CD39 expression correlates with exposure to hypoxia and Texh sorted from tumors engineered to be less hypoxic displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, contributes to generation of immunosuppressive adenosine, and has been previously associated with terminal exhaustion.11–13 Conclusions Our data support a model that as CTL progress to terminal exhaustion, hypoxic exposure enforces the upregulation of CD39, providing Texh a mechanism to suppress proinflammatory processes. These findings suggest Texh are not solely dysfunctional but rather are deleterious to anti-tumor immunity and may need to be drastically reprogrammed or deleted in order to alleviate immunosuppressive functions. References Wolchok JD. et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N. Engl. J. Med 2017; 377, 1345–1356. Hellmann MD, et al. Nivolumab plus ipilimumab as first-line treatment for advanced non-small-cell lung cancer (CheckMate 012): results of an open-label, phase 1, multicohort study. Lancet Oncol 2017; 18, 31–41. Robert C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 2015; 372, 2521–2532. Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat. Immunol 2019;20:326–336. Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421. Blackburn SD, Shin H, Freeman GJ & Wherry EJ. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc. Natl. Acad. Sci 2008;105:15016–15021. Pauken KE, et al. Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science 2016;354:1160–1165. Najjar YG, et al. Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma. JCI Insight. 2019; 4. Loo K, et al. Partially exhausted tumor-infiltrating lymphocytes predict response to combination immunotherapy. JCI Insight 2017; 2. Daud AI, et al. Tumor immune profiling predicts response to anti-PD-1 therapy in human melanoma. J. Clin. Invest 2016;126:3447–3452. . Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun 2018;9:2724. Canale FP, et al. CD39 Expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128. Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11, e1005177.

Tumor-infiltrating T cells experience metabolic repression that hinders their function and thus response to immunotherapy. Menk et al. show that ligation of 4-1BB, a TNFR family costimulatory molecule, can promote increased metabolic sufficiency, which enables intratumoral T cell function and response to immunotherapy., Despite remarkable responses to cancer immunotherapy in a subset of patients, many patients remain resistant to these therapies. The tumor microenvironment can impose metabolic restrictions on T cell function, creating a resistance mechanism to immunotherapy. We have previously shown tumor-infiltrating T cells succumb to progressive loss of metabolic sufficiency, characterized by repression of mitochondrial activity that cannot be rescued by PD-1 blockade. 4-1BB, a costimulatory molecule highly expressed on exhausted T cells, has been shown to influence metabolic function. We hypothesized that 4-1BB signaling might provide metabolic support to tumor-infiltrating T cells. 4-1BB costimulation of CD8+ T cells results in enhanced mitochondrial capacity (suggestive of fusion) and engages PGC1α-mediated pathways via activation of p38-MAPK. 4-1BB treatment of mice improves metabolic sufficiency in endogenous and adoptive therapeutic CD8+ T cells. 4-1BB stimulation combined with PD-1 blockade results in robust antitumor immunity. Sequenced studies revealed the metabolic support afforded by 4-1BB agonism need not be continuous and that a short course of anti–4-1BB pretreatment was sufficient to provide a synergistic response. Our studies highlight metabolic reprogramming as the dominant effect of 4-1BB therapy and suggest that combinatorial strategies using 4-1BB agonism may help overcome the immunosuppressive metabolic landscape of the tumor microenvironment., Graphical Abstract

BackgroundOncolytic viruses are an underappreciated immunotherapy capable of inflaming the tumor microenvironment (TME), vaccinating a patient against their own tumor, and delivering gene therapy to the TME. However, apart from the oncolytic HSV T-vec, these therapies have not seen widespread use, due in part to incomplete understanding of their immunologic mechanisms of action. We sought to determine features of oncolytic vaccinia virus (VV) response and resistance using subclones of the HPV+ head and neck cancer model MEER rendered sensitive or resistant to VV.MethodsA VV sensitive MEER tumor resisting treatment was serially passaged in mice and treated with VV until a stably resistant line was generated (Fig1). Sensitive or resistant MEER tumors were implanted, treated with a single intratumoral dose of VV, and harvested 4–7 days later for cytometric analysis. A genetically encoded TGFβ inhibitor was recombined into oncolytic VV (VV-TGFβi).ResultsWe used serial in vivo passaging to generate a VV-resistant MEER line (MEERvvR) from one sensitive to VV (MEERvvS, figure 1) and compared their immune infiltrate. While VV promoted acute cytokine production and cytotoxicity in conventional T cells, the major determining factor between sensitivity and resistance was the phenotype of Treg cells. At baseline, Treg cells in MEERvvS had lower Nrp1 expression and higher IFNγ-STAT1 signaling compared to MEERvvR, indicative of Treg 'fragility'. VV treatment induced MEERvvS Treg cells to become immunostimulatory and produce IFNγ (figure 2). RNAseq revealed MEERvvR produced more TGFβ than MEERvvS cells, suggesting these tumors directly stabilize Treg cells. To determine if MEERvvR could be sensitized to VV, we engineered oncolytic vaccinia to produce a genetically-encoded TGFβ inhibitor which binds TGFβRII, preventing TGFβ1-3 binding (VV-TGFβi). When MEERvvR were treated with VV-TGFβi, elite responses were restored, with commensurate increase in survival (figure 3) associated with increased STAT1 signaling in Treg cells.ConclusionsResistance to oncolytic vaccinia is controlled by Treg cell phenotype; tumors harboring more fragile Treg cells respond exquisitely to VV. An oncolytic vaccinia engineered to produce a novel TGFβi could remodel the TME to be less supportive of Tregs, rendering resistant tumors sensitive to VV. Our data highlight the importance of Treg cell status in resistance to oncolytic virus therapy and suggest TGFβ can be effectively targeted through an inhibitor encoded within the virus. Importantly, this TME directed production of the TGFβi carries no toxicity previously associated with systemic TGFβ inhibition, suggesting a viral approach to TGFβ inhibition can be an effective strategy support broader immunotherapy response.Abstract 743 Figure 1Strategy used to generate a vaccinia resistant MEER (MEERvvR) from vaccinia sensitive MEER (MEERvvS)Abstract 743 Figure 2IFNγ production in Treg cells in MEERvvS and MEERvvR after treatment with PBS or control vaccinia (VV-Ctrl)Abstract 743 Figure 3Survival of VV-resistant MEER treated with PBS, control vaccinia (VV-Ctrl), or vaccinia engineered to deliver a potent inhibitor of TGFβ (VV-TGFβi)

BackgroundWhile CD8+ cytotoxic T cells are clearly critical for identification and elimination of cancer cells, factors concentrated within the tumor microenvironment drive altered differentiation of these cells to a hypofunctional, short-lived state termed T cell exhaustion1 (figure 1a). Exhaustion is a progressive lineage, and it is now clear that terminally exhausted T (tTexh) cells are not the targets of checkpoint blockade immunotherapy but may serve as factors that limit immunotherapeutic efficacy.2–6 Compared directly, tumor-infiltrating CD8+ tTexh cells bear notable phenotypic similarity to CD4+Foxp3+ regulatory T (Treg) cells in expression of immunosuppressive molecules suggesting beyond loss of effector function, tTexh cells may be directly anti-functional and constrain tumor-specific immunity. Thus, we hypothesize that tTexh cells potentiate the suppressive microenvironment of solid tumor and that strategies to limit their generation or reprogram their immunosuppressive nature will improve control of tumor progression.MethodsT cell populations were isolated from murine tumor lines, B16-F10 melanoma, Ptenflox/floxBrafLSL.V600ETyr2Cre.ERT2–derived Clone 24 melanoma, MEER head and neck carcinoma, and MC38 adenocarcinoma. T cell-specific CD39 (Entpd1) deletion was accomplished by crossing Entpd1flox/flox mice to Cd4Cre or E8iGFP-Cre-ERT2. Enforced expression of CD39 in effector T cells was attained by murine retroviral vector delivery. Tumor hypoxia was alleviated by CRISPR-Cas9-directed deletion of mitochondrial genes in B16-F10 or by treatment with axitinib or metformin.ResultsWhen sorted directly from tumor, CD8+PD-1hiTim-3+ tTexh cells, but not progenitor PD-1intTim-3– pTexh cells, induce marked suppression of T cell effector responses, comparable to CD4+Foxp3+ Treg cells from the same environment (figure 1b-c). The ectonucleotidase, CD39, increases as cells progressively differentiate and is associated with terminal exhaustion.7 8 CD8+ T cell-restricted deletion of CD39 restricts regulatory functions of tTexh cells (figure 1b), improving tumor control and augmenting response to checkpoint blockade (figure 1d). CD39 expression correlates with hypoxia exposure and tTexh cells sorted from tumors engineered to be less hypoxic or treated with hypoxia-mitigating agents displayed a significant loss of suppressive capacity. Our data suggest that tumor hypoxia enforces Hif1a-dependent expression of CD39 which depletes extracellular ATP, supports adenosine generation, and limits therapeutic efficacy.ConclusionsOur data support a model that as CD8+ T cells progress to terminal exhaustion, hypoxia exposure enforces the upregulation of CD39, providing tTexh cells a mechanism to suppress proinflammatory processes and promote tumor progression. These findings suggest tTexh cells are not solely dysfunctional but rather are deleterious to antitumor immunity and may need to be drastically reprogrammed or depleted to improve patient outcomes.ReferencesBlank CU, et al. Defining “T cell exhaustion”. Nat Rev Immunol 2019;19:665–674.Miller BC, et al. Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade. Nat Immunol 2019;20:326–336.Blackburn SD, et al. Selective expansion of a subset of exhausted CD8 T cells by alphaPD-L1 blockade. Proc Natl Acad Sci USA 2008;105:15016–15021.Sade-Feldman M, et al. Defining T Cell states associated with response to checkpoint immunotherapy in Melanoma. Cell 2018;175:998–1013.e20.Im SJ, et al. Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy. Nature 2016;537:417–421.Siddiqui I, et al. Intratumoral Tcf1+PD-1+CD8+ T Cells with stem-like properties promote tumor control in response to vaccination and checkpoint blockade immunotherapy. Immunity 2019;50:195–211.e10.Canale FP, et al. CD39 expression defines cell exhaustion in tumor-infiltrating CD8+ T Cells. Cancer Res 2018;78:115–128.Gupta PK, et al. CD39 expression identifies terminally exhausted CD8+ T cells. PLoS Pathog 2015;11:e1005177.Abstract 679 Figure 1(a) Schematic depicting differentiation of CD8+ T cells to terminal exhaustion in cancer and subsequent suppression of local immune responses by expression of ectonucleosidase, CD39; (b) When assayed directly ex vivo, CD8+ terminally exhausted T (tTexh) cells, but not progenitor exhausted T (pTexh) cells, suppress effector functions as effectively as CD4+Foxp3+ Treg isolated from the same environment. Deletion of CD39 alleviates tTexh-mediated suppression; (c) CD8+ T cell suppression correlates with expression of CD39 on tTexh from various tumor models. (d) CD8+ T cell-specific deletion of CD39 slows tumor growth and improves immune response to checkpoint blockade-resistant tumors. Data are pooled from ≥3 experiments. Statistics are two-way ANOVA with multiple comparisons or Pearson correlation. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Many genes aberrantly expressed in prostate cancer are involved in normal basal to luminal cell differentiation. We previously demonstrated that transient ING4 expression is required for luminal cell differentiation and is downregulated in ~60% of primary prostate tumors. We further demonstrated in a primary prostate cancer model overexpressing ERG, MYC, and shPTEN (EMP) that loss of PTEN was responsible for ING4 loss. Furthermore, half of the human tumor samples that lose ING4 have also lost PTEN. However, we did not know how PTEN loss inhibits ING4 expression. Utilizing our in vitro differentiation model, whereby prostate basal epithelial cells (iPrEC) treated with KGF and androgen induce a suprabasal layer of luminal-like cells, and RNA-seq we identified transcriptional nodes required for luminal differentiation. Differentially expressed genes were analyzed by GeneGo to identify enriched transcription-factor signatures. Of the ~600 differentially regulated genes during differentiation, the largest signature (29% of genes) was CREB/ATF targets. Induction of Blimp1, Claudin1, and Plk2 and inhibition of Chek1 were further validated by qRT-PCR and immunoblotting. CREB/ATF bind constitutively to open chromatin CRE elements in the promoters of genes and are activated through signaling-induced phosphorylation at Ser133 by kinases, including AKT. We found that both CREB1 and ATF1 are inducibly phosphorylated midway through luminal differentiation, with ATF1 preceding CREB1. Knockdown of CREB1 with shRNA increased ING4, accelerated differentiation, and induced premature luminal cell death. Conversely, knockdown of ATF1 blocked ING4 induction and prevented suprabasal layer formation. CREB1/ATF1 ChIP was enriched at the ING4 promoter at mid-differentiation, when ING4 expression peaks. Additionally, CREB1/ATF1 was constitutively bound to the promoter of JFK, an E3-ligase that targets ING4 and whose mRNA levels increase during differentiation. Thus, we propose that ATF1 is required to induce ING4 transcription, while CREB1 suppresses ING4 and simultaneously activates its E3-ligase to tightly control the timing of ING4 expression. We compared the gene signature of luminal cells to that of the tumorigenic EMP cells and, surprisingly, found that 30% of the differentially expressed genes were also CREB/ATF targets. However, there is less than 10% overlap in these targets, indicating that CREB/ATF control distinct subsets of genes in differentiated luminal cells versus cancer cells. Some of EMP-specific CREB/ATF targets included GATA2, TWIST1, Necdin, and PPM1F, which were further validated by qRT-PCR and immunoblotting. CREB1 and ATF1 were highly phosphorylated in EMP cells and knockdown of CREB1 restored ING4 expression and suprabasal formation. Our working model is that AKT activation upon PTEN loss in transiently differentiating luminal cells results in premature and constitutive activation of CREB1/ATF1 bound to genes prior to induction of the ING4 chromatin switch. This prevents ING4 induction and the chromatin rearrangements required for terminal differentiation. In normal PrECs, CREB/ATF1 activation is tightly controlled by as yet undetermined factors and is only permitted when the proper CRE binding sites are exposed. This model helps to explain how loss of PTEN disrupts luminal cell terminal differentiation to promote prostate cancer oncogenesis. Citation Format: McLane Watson, Penny Berger, Sander Frank, Mary Winn, Cindy Miranti. CREB1 and ATF1 differentially regulate terminal prostate luminal differentiation by controlling the timing of ING4 expression, while CREB1 prevents ING4 expression upon PTEN loss in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B031.

Asymmetric cell division is critical during development, as it influences processes such as cell fate specification and cell migration. We have characterized FRK-1, a homolog of the mammalian Fer nonreceptor tyrosine kinase, and found it to be required for differentiation and maintenance of epithelial cell types, including the stem cell-like seam cells of the hypodermis. A genomic knockout of frk-1, allele ok760, results in severely uncoordinated larvae that arrest at the L1 stage and have an excess number of lateral hypodermal cells that appear to have lost asymmetry in the stem cell-like divisions of the seam cell lineage. frk-1(ok760) mutants show that there are excess lateral hypodermal cells that are abnormally shaped and smaller in size compared to wild type, a defect that could be rescued only in a manner dependent on the kinase activity of FRK-1. Additionally, we observed a significant change in the expression of heterochronic regulators in frk-1(ok760) mutants. However, frk-1(ok760) mutants do not express late, nonseam hypodermal GFP markers, suggesting the seam cells do not precociously differentiate as adult-hyp7 cells. Finally, our data also demonstrate a clear role for FRK-1 in seam cell proliferation, as eliminating FRK-1 during the L3–L4 transition results in supernumerary seam cell nuclei that are dependent on asymmetric Wnt signaling. Specifically, we observe aberrant POP-1 and WRM-1 localization that is dependent on the presence of FRK-1 and APR-1. Overall, our data suggest a requirement for FRK-1 in maintaining the identity and proliferation of seam cells primarily through an interaction with the asymmetric Wnt pathway.

Overexpression of Myc and loss of Pten are very common oncogenic events in prostate cancer. However, why these specific oncogenic events, as opposed to others, are involved in prostate cancer develop remains unclear. While the impact of Myc and Pten on cell proliferation is well-characterized, their role in differentiation and the resulting impact on tumorigenesis is poorly understood. We recently developed an in vitro differentiation model in which basal prostate epithelial cells (PrECs) can be differentiated into secretory luminal cells. Our model offers several advantages, in that it is human-specific and readily manipulated genetically and biochemically; providing a rare opportunity to dissect the specific genetic and biochemical events associated with human PrEC differentiation and determining how it is impacted by specific oncogenic events. Using this model, we tested the hypothesis that overexpression of Myc and loss of Pten induce prostate cancer because their dysregulation impairs differentiation of an intermediate progenitor cell population that marks the tumor cell of origin. Transient induction of both Pten and Myc are required for normal PrEC differentiation. Constitutive Myc overexpression initially accelerates differentiation, but the differentiated cells ultimately die via a p53-independent mechanism. Overexpression of Myc in combination with loss of Pten (Myc+shPten), is required for tumorigenesis because it rescues cell survival and blocks terminal differentiation. We identified the chromatin binding protein, ING4, as a Myc target required for PrEC terminal differentiation. ING4 is lost in over 60% of human prostate tumors and in the Myc+shPten cells, ING4 expression is blocked. Loss of ING4 in Myc overexpressing cells is sufficient to replace loss of Pten; ING4 loss, like Pten loss, is required for tumorigenesis to block terminal differentiation and keep cells alive. We further identified Miz1, a component of the Myc repressor complex, as a direct target of ING4 required for terminal PrEC differentiation. Myc/Miz1 is required to suppress integrin α6 and β1 expression during terminal PrEC differentiation; in Myc+shPten cells, Miz1 fails to be induced and integrin α6β1 is aberrantly co-expressed with AR in an intermediate progenitor and tumorigenic cell population. Conclusions: Prostate cancer oncogenesis requires dysregulation of Myc and Pten, because they are required for the normal PrEC terminal differentiation pathway. Pten loss is required to remove the ING4/Miz1-mediated terminal differentiation program initiated by Myc overexpression, to maintain the intermediate progenitor cells in a partially differentiated, yet proliferative state, and to evade cell death. Citation Format: Penny Berger, McLane Watson, Mary Winn, Cindy K. Miranti. Key intermediate progenitor in luminal prostate epithelial differentiation dictates susceptibility to Myc overexpression and Pten loss in prostate cancer cell of origin. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B20.

Limiting metabolic competition in the tumour microenvironment may increase the effectiveness of immunotherapy. Owing to its crucial role in the glucose metabolism of activated T cells, CD28 signalling has been proposed as a metabolic biosensor of T cells 1 . By contrast, the engagement of CTLA-4 has been shown to downregulate T cell glycolysis 1 . Here we investigate the effect of CTLA-4 blockade on the metabolic fitness of intra-tumour T cells in relation to the glycolytic capacity of tumour cells. We found that CTLA-4 blockade promotes metabolic fitness and the infiltration of immune cells, especially in glycolysis-low tumours. Accordingly, treatment with anti-CTLA-4 antibodies improved the therapeutic outcomes of mice bearing glycolysis-defective tumours. Notably, tumour-specific CD8 + T cell responses correlated with phenotypic and functional destabilization of tumour-infiltrating regulatory T (T reg ) cells towards IFNγ- and TNF-producing cells in glycolysis-defective tumours. By mimicking the highly and poorly glycolytic tumour microenvironments in vitro, we show that the effect of CTLA-4 blockade on the destabilization of T reg cells is dependent on T reg cell glycolysis and CD28 signalling. These findings indicate that decreasing tumour competition for glucose may facilitate the therapeutic activity of CTLA-4 blockade, thus supporting its combination with inhibitors of tumour glycolysis. Moreover, these results reveal a mechanism by which anti-CTLA-4 treatment interferes with T reg cell function in the presence of glucose.