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α2δ-1 (encoded by theCacna2d1gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at theCacna2d1promoter in the DRG. Strikingly,Hdac2knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1–NMDA receptor interaction at the spinal cord level.Hdac2deletion in DRG neurons increased histone acetylation levels at theCacna2d1promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1–dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly,Hdac2knockdown-induced pain hypersensitivity was blunted inCacna2d1knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at theCacna2d1promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENTExcess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

Tumor angiogenesis is a cancer hallmark, and its therapeutic inhibition has provided meaningful, albeit limited, clinical benefit. While anti-angiogenesis inhibitors deprive the tumor of oxygen and essential nutrients, cancer cells activate metabolic adaptations to diminish therapeutic response. Despite these adaptations, angiogenesis inhibition incurs extensive metabolic stress, prompting us to consider such metabolic stress as aninduced vulnerabilityto therapies targeting cancer metabolism. Metabolomic profiling of angiogenesis-inhibited intracranial xenografts showed universal decrease in tricarboxylic acid cycle intermediates, corroborating a state of anaplerotic nutrient deficit or stress. Accordingly, we show strong synergy between angiogenesis inhibitors (Avastin, Tivozanib) and inhibitors of glycolysis or oxidative phosphorylation through exacerbation of anaplerotic nutrient stress in intracranial orthotopic xenografted gliomas. Our findings were recapitulated in GBM xenografts that do not have genetically predisposed metabolic vulnerabilities at baseline. Thus, our findings cement the central importance of the tricarboxylic acid cycle as the nexus of metabolic vulnerabilities and suggest clinical path hypothesis combining angiogenesis inhibitors with pharmacological cancer interventions targeting tumor metabolism for GBM tumors.

Supplementary Data from Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

Supplementary Table from Synthetic Essentiality of Tryptophan 2,3-Dioxygenase 2 in APC-Mutated Colorectal Cancer

Inactivation of adenomatous polyposis coli (APC) is common across many cancer types and serves as a critical initiating event in most sporadic colorectal cancers. APC deficiency activates WNT signaling, which remains an elusive target for cancer therapy, prompting us to apply the synthetic essentiality framework to identify druggable vulnerabilities for APC-deficient cancers. Tryptophan 2,3-dioxygenase 2 (TDO2) was identified as a synthetic essential effector of APC-deficient colorectal cancer. Mechanistically, APC deficiency results in the TCF4/β-catenin–mediated upregulation of TDO2 gene transcription. TDO2 in turn activates the Kyn–AhR pathway, which increases glycolysis to drive anabolic cancer cell growth and CXCL5 secretion to recruit macrophages into the tumor microenvironment. Therapeutically, APC-deficient colorectal cancer models were susceptible to TDO2 depletion or pharmacologic inhibition, which impaired cancer cell proliferation and enhanced antitumor immune profiles. Thus, APC deficiency activates a TCF4–TDO2–AhR–CXCL5 circuit that affects multiple cancer hallmarks via autonomous and nonautonomous mechanisms and illuminates a genotype-specific vulnerability in colorectal cancer.Significance:This study identifies critical effectors in the maintenance of APC-deficient colorectal cancer and demonstrates the relationship between APC/WNT pathway and kynurenine pathway signaling. It further determines the tumor-associated macrophage biology in APC-deficient colorectal cancer, informing genotype-specific therapeutic targets and the use of TDO2 inhibitors.This article is highlighted in the In This Issue feature, p. 1599

Figure S1 shows that CLOCK/BMAL1 is essential for GSC self-­renewal, but not required for glioma cell proliferation.

Supplementary Figure S1. CyTOF analysis of biological samples from Ptenpc-/-Smad4pc-/- mice (Related to Figure 2). Supplementary Figure S2. Strategy used for MDSCs Isolation (Related to Figure 3). Supplementary Figure S3. Treatment scheme for Gr-1 antibody, peptibody, and Cxcr2 inhibitor SB225002. Supplementary Figure S4. IHC staining of Ki67, CD45, Vimentin, Smooth muscle actin (SMA) and Trichrome staining of mouse prostate tissues treated with IgG control or Gr1 antibody. Supplementary Figure S5. The top 10 differentially expressed genes in Ptenpc-/-Smad4pc-/- tumors as compared to Ptenpc-/- tumors, identified by microarray analysis (n=5). Figure S6. Top 10 activated oncogenic signatures identified by GSEA analysis in Ptenpc-/- Smad4pc-/- tumors as compared to Ptenpc-/- tumors (n=5). Figure S7. Clustering of primary prostate tumors from Wallace et al into MDSC-high and MDSC-low subtypes.

Overlapped genes between Genes upregulated in Ptenpc-/-Smad4pc-/- tumors as compared to Ptenpc-/- tumors ({greater than or equal to}2 fold) and genes upregulated in GFP+ tumors cells from Ptenpc-/-Smad4pc-/- mice as compared to Tomato+ cells ({greater than or equal to}4 fold).

Subject demographics in the University of Michigan sample set

Oncogenic KRAS (KRAS*) is a key tumor maintenance gene in pancreatic ductal adenocarcinoma (PDAC), motivating pharmacologic targeting of KRAS* and its effectors. Here, we explored mechanisms involving the tumor microenvironment (TME) as a potential basis for resistance to targeting KRAS*. Using the inducible KrasG12D;Trp53−/− PDAC mouse model, gain-of-function screens of epigenetic regulators identified HDAC5 as the top hit enabling KRAS* independent tumor growth. HDAC5-driven escaper tumors showed a prominent neutrophil-to-macrophage switch relative to KRAS*-driven tumors. Mechanistically, HDAC5 represses Socs3, a negative regulator of chemokine CCL2, resulting in increased CCL2, which recruits CCR2+ macrophages. Correspondingly, enforced Ccl2 promotes macrophage recruitment into the TME and enables tumor recurrence following KRAS* extinction. These tumor-associated macrophages in turn provide cancer cells with trophic support including TGFβ to enable KRAS* bypass in a SMAD4-dependent manner. Our work uncovers a KRAS* resistance mechanism involving immune cell remodeling of the PDAC TME.Significance:Although KRAS* is required for PDAC tumor maintenance, tumors can recur following KRAS* extinction. The capacity of PDAC cancer cells to alter the TME myeloid cell composition to support KRAS*-independent tumor growth illuminates novel therapeutic targets that may enhance the effectiveness of therapies targeting KRAS* and its pathway components.See related commentary by Carr and Fernandez-Zapico, p. 910.This article is highlighted in the In This Issue feature, p. 890

A hallmark of pancreatic ductal adenocarcinoma (PDAC) is an exuberant stroma comprised of diverse cell types that enable or suppress tumor progression. Here, we explored the role of oncogenic KRAS in protumorigenic signaling interactions between cancer cells and host cells. We show that KRAS mutation (KRAS*) drives cell-autonomous expression of type I cytokine receptor complexes (IL2rγ–IL4rα and IL2rγ–IL13rα1) in cancer cells that in turn are capable of receiving cytokine growth signals (IL4 or IL13) provided by invading Th2 cells in the microenvironment. Early neoplastic lesions show close proximity of cancer cells harboring KRAS* and Th2 cells producing IL4 and IL13. Activated IL2rγ–IL4rα and IL2rγ–IL13rα1 receptors signal primarily via JAK1–STAT6. Integrated transcriptomic, chromatin occupancy, and metabolomic studies identified MYC as a direct target of activated STAT6 and that MYC drives glycolysis. Thus, paracrine signaling in the tumor microenvironment plays a key role in the KRAS*-driven metabolic reprogramming of PDAC.Significance:Type II cytokines, secreted by Th2 cells in the tumor microenvironment, can stimulate cancer cell–intrinsic MYC transcriptional upregulation to drive glycolysis. This KRAS*-driven heterotypic signaling circuit in the early and advanced tumor microenvironment enables cooperative protumorigenic interactions, providing candidate therapeutic targets in the KRAS* pathway for this intractable disease.

Normalized spectra counts of AK1, CLIC1, and SOD1 in eight CRC cell lines

Case/control ratios of 22 proteins selected for antibody microarrays in pre-diagnostic CRC plasma samples from the Women's Health Initiative cohort

Glioblastoma (GBM) is a lethal brain tumor containing a subpopulation of glioma stem cells (GSC). Pan-cancer analyses have revealed that stemness of cancer cells correlates positively with immunosuppressive pathways in many solid tumors, including GBM, prompting us to conduct a gain-of-function screen of epigenetic regulators that may influence GSC self-renewal and tumor immunity. The circadian regulator CLOCK emerged as a top hit in enhancing stem-cell self-renewal, which was amplified in about 5% of human GBM cases. CLOCK and its heterodimeric partner BMAL1 enhanced GSC self-renewal and triggered protumor immunity via transcriptional upregulation of OLFML3, a novel chemokine recruiting immune-suppressive microglia into the tumor microenvironment. In GBM models, CLOCK or OLFML3 depletion reduced intratumoral microglia density and extended overall survival. We conclude that the CLOCK–BMAL1 complex contributes to key GBM hallmarks of GSC maintenance and immunosuppression and, together with its downstream target OLFML3, represents new therapeutic targets for this disease.Significance:Circadian regulator CLOCK drives GSC self-renewal and metabolism and promotes microglia infiltration through direct regulation of a novel microglia-attracting chemokine, OLFML3. CLOCK and/or OLFML3 may represent novel therapeutic targets for GBM.This article is highlighted in the In This Issue feature, p. 327

Table S1 is a list of primers used for qRT-­PCR analysis.

Supplementary Figures 1-6

Epigenetic library information

Supplementary Tables 1-2, Figures 1-8 from PTEN Is a Major Tumor Suppressor in Pancreatic Ductal Adenocarcinoma and Regulates an NF-κB–Cytokine Network

Supplementary Methods

Normalized intensity of high density antibody array and CEA levels in the University of Michigan sample set

MAPRE1 protein expression in tissue microarray

Inactivation of adenomatous polyposis coli (APC) is common across many cancer types and serves as a critical initiating event in most sporadic colorectal cancers. APC deficiency activates WNT signaling, which remains an elusive target for cancer therapy, prompting us to apply the synthetic essentiality framework to identify druggable vulnerabilities for APC-deficient cancers. Tryptophan 2,3-dioxygenase 2 (TDO2) was identified as a synthetic essential effector of APC-deficient colorectal cancer. Mechanistically, APC deficiency results in the TCF4/β-catenin–mediated upregulation of TDO2 gene transcription. TDO2 in turn activates the Kyn–AhR pathway, which increases glycolysis to drive anabolic cancer cell growth and CXCL5 secretion to recruit macrophages into the tumor microenvironment. Therapeutically, APC-deficient colorectal cancer models were susceptible to TDO2 depletion or pharmacologic inhibition, which impaired cancer cell proliferation and enhanced antitumor immune profiles. Thus, APC deficiency activates a TCF4–TDO2–AhR–CXCL5 circuit that affects multiple cancer hallmarks via autonomous and nonautonomous mechanisms and illuminates a genotype-specific vulnerability in colorectal cancer. Significance: This study identifies critical effectors in the maintenance of APC-deficient colorectal cancer and demonstrates the relationship between APC/WNT pathway and kynurenine pathway signaling. It further determines the tumor-associated macrophage biology in APC-deficient colorectal cancer, informing genotype-specific therapeutic targets and the use of TDO2 inhibitors. This article is highlighted in the In This Issue feature, p. 1599

E26 transformation-specific transcription factor ERG is aberrantly overexpressed in approximately 50% of all human prostate cancer due to TMPRSS2-ERG gene rearrangements. However, mice with prostate-specific transgenic expression of prostate cancer–associated ERG alone fail to develop prostate cancer, highlighting that ERG requires other lesions to drive prostate tumorigenesis. Forkhead box (FOXO) transcription factor FOXO1 is a tumor suppressor that is frequently inactivated in human prostate cancer. Here, we demonstrate that FOXO1, but not other FOXO proteins (FOXO3 and FOXO4), binds and inhibits the transcriptional activity of prostate cancer–associated ERG independently of FOXO1 transcriptional activity. Knockdown of endogenous FOXO1 increased invasion of TMPRSS2–ERG fusion–positive VCaP cells, an effect completely abolished by ERG knockdown. Patient specimen analysis demonstrated that FOXO1 and ERG protein expression inversely correlated in a subset of human prostate cancer. Although human ERG transgene expression or homozygous deletion of Foxo1 alone in the mouse prostate failed to promote tumorigenesis, concomitant ERG transgene expression and Foxo1 deletion resulted in upregulation of ERG target genes, increased cell proliferation, and formation of high-grade prostatic intraepithelial neoplasia. Overall, we provide biochemical and genetic evidence that aberrantly activated ERG cooperates with FOXO1 deficiency to promote prostate tumorigenesis and cell invasion. Our findings enhance understanding of prostate cancer etiology and suggest that the FOXO1–ERG signaling axis can be a potential target for treatment of prostate cancer. Cancer Res; 77(23); 6524–37. ©2017 AACR.

Different inhibitors of endocytosis

Supplementary Information. File containing Materials and Methods, references for Materials and Methods, a table of PCR primers for genotyping and RT-qPCR studies, and Supplementary Figure legends.

Supplementary Figure S2. In panel A, immunohistochemistry for ERG in wild-type, Foxo1pc-/-, Pb-ERG and Foxo1pc-/-; Pb-ERG mouse prostates. In panel B, immunohistochemistry for FOXO1 in wild-type, Foxo1pc-/-, Pb-ERG and Foxo1pc-/-; Pb-ERG mouse prostates. ERG and FOXO1 are localized to the nucleus in mice where expression is expected.

Supplementary Data from Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma

Altered metabolism helps sustain cancer cell proliferation and survival. Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (PKM2), which can support anabolic metabolism to support cell proliferation. However, Pkm2 expression is dispensable for the formation and growth of many cancers in vivo. Expression of pyruvate kinase isoform M1 (Pkm1) is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of PKM1 in cancer has been less studied than PKM2. To test how differential expression of pyruvate kinase isoforms affects cancer initiation and progression, we generated mice harboring a conditional allele of Pkm1 and crossed these mice, or those with a Pkm2 conditional allele, with a Pten loss-driven prostate cancer model. Pkm1 loss led to increased PKM2 expression and accelerated prostate cancer development, whereas Pkm2 deletion led to increased PKM1 expression and suppressed tumor progression. Metabolic profiling revealed altered nucleotide levels in tumors with high PKM1 expression, and failure of these tumors to progress was associated with DNA replication stress and senescence. Consistent with these data, a small molecule pyruvate kinase activator that mimics a high activity PKM1-like state suppressed progression of established prostate tumors. Analysis of human specimens showed PKM2 expression is retained in most human prostate cancers. Overall, this study uncovers a role for pyruvate kinase isoforms in prostate cancer initiation and progression, and argues that pharmacologic pyruvate kinase activation may be beneficial for treating prostate cancer.Significance:Differential expression of PKM1 and PKM2 impacts prostate tumorigenesis and suggests a potential therapeutic vulnerability in prostate cancer.

Supplementary Table from Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma

Supplementary Figure from Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma

Purpose:Prostate cancers show remarkable resistance to emerging immunotherapies, partly due to tolerogenic STAT3 signaling in tumor-associated myeloid cells. Here, we describe a novel strategy combining STAT3 inhibition with Toll-like Receptor 9 (TLR9) stimulation to unleash immune response against prostate cancers regardless of the genetic background.Experimental Design:We developed and validated a conjugate of the STAT3 antisense oligonucleotide (ASO) tethered to immunostimulatory TLR9 agonist (CpG oligonucleotide) to improve targeting of human and mouse prostate cancer and myeloid immune cells, such as myeloid-derived suppressor cells (MDSC).Results:CpG-STAT3ASO conjugates showed improved biodistribution and potency of STAT3 knockdown in target cells in vitro and in vivo. Systemic administration of CpG-STAT3ASO (5 mg/kg) eradicated bone-localized, Ras/Myc-driven, and Ptenpc−/−Smad4pc−/−Trp53c−/− prostate tumors in the majority of treated mice. These antitumor effects were primarily immune-mediated and correlated with an increased ratio of CD8+ to regulatory T cells and reduced pSTAT3+/PD-L1+ MDSCs. Both innate and adaptive immunity contributed to systemic antitumor responses as verified by the depletion of Gr1+ myeloid cells and CD8+ and CD4+ T cells, respectively. Importantly, only the bifunctional CpG-STAT3ASO, but not control CpG oligonucleotides, STAT3ASO alone, or the coinjection of both oligonucleotides, succeeded in recruiting neutrophils and CD8+ T cells into tumors. Thus, the concurrence of TLR9 activation with STAT3 inhibition in the same cellular compartment is indispensable for overcoming tumor immune tolerance and effective antitumor immunity against prostate cancer.Conclusions:The bifunctional, immunostimulatory, and tolerance-breaking design of CpG-STAT3ASO offers a blueprint for the development of effective and safer oligonucleotide strategies for treatment of immunologically “cold” human cancers.

Supplementary Table 2 from Common and Distinct Genomic Events in Sporadic Colorectal Cancer and Diverse Cancer Types

Supplementary Figure 2 from Common and Distinct Genomic Events in Sporadic Colorectal Cancer and Diverse Cancer Types

Supplementary Figures 1-5 from A Genome-Wide Screen Reveals Functional Gene Clusters in the Cancer Genome and Identifies EphA2 as a Mitogen in Glioblastoma

Supplementary Figure 3 from Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes

Mutated KRAS (KRAS*) is a fundamental driver in the majority of pancreatic ductal adenocarcinomas (PDAC). Using an inducible mouse model of KRAS*-driven PDAC, we compared KRAS* genetic extinction with pharmacologic inhibition of MEK1 in tumor spheres and in vivo. KRAS* ablation blocked proliferation and induced apoptosis, whereas MEK1 inhibition exerted cytostatic effects. Proteomic analysis evidenced that MEK1 inhibition was accompanied by a sustained activation of the PI3K–AKT–MTOR pathway and by the activation of AXL, PDGFRa, and HER1–2 receptor tyrosine kinases (RTK) expressed in a large proportion of human PDAC samples analyzed. Although single inhibition of each RTK alone or plus MEK1 inhibitors was ineffective, a combination of inhibitors targeting all three coactivated RTKs and MEK1 was needed to inhibit proliferation and induce apoptosis in both mouse and human low-passage PDAC cultures. Importantly, constitutive AKT activation, which may mimic the fraction of AKT2-amplified PDAC, was able to bypass the induction of apoptosis caused by KRAS* ablation, highlighting a potential inherent resistance mechanism that may inform the clinical application of MEK inhibitor therapy. This study suggests that combinatorial-targeted therapies for pancreatic cancer must be informed by the activation state of each putative driver in a given treatment context. In addition, our work may offer explanative and predictive power in understanding why inhibitors of EGFR signaling fail in PDAC treatment and how drug resistance mechanisms may arise in strategies to directly target KRAS. Cancer Res; 75(6); 1091–101. ©2015 AACR.

supplementary Figures S1-S10. S1 growth and regression of iKRAS tumors. S2 culture comparison. S3 immunohistochemistry of p-erk and p-s6 S4 Phospho rtk array S5 Validation of anti AXL antibodies S6 validationn of the AXL inhibitor in vitro S7 validationn of the AXL inhibitor in vivo S8 efficacy of targeting AXL S9 RTK array of iKRAS cells treated with multiple RTK inhibitors S10 P-ERK levels

Supplementary Figure 6 from Antitumor Activity of a Small-Molecule Inhibitor of Human Silent Information Regulator 2 Enzymes