Objectives: This study investigates the relationship between serum oxylipin profiles and response to immune checkpoint inhibitor therapy in melanoma subtypes to identify potential metabolic biomarkers for treatment response. Methods: In a retrospective cohort study, serum samples from 43 stage III and stage IV melanoma patients treated at the University of Colorado Hospital from 2010 to 2023 were analyzed via ultra-high-pressure liquid chromatography-mass spectrometry. Melanoma patients were treated anti-PD-1 monotherapy or combination immune checkpoint inhibitor therapy and response was assessed using RECIST 1.1 criteria. Results: Using mass spectroscopy, we determined global oxylipin metabolite profiles are largely uniform pre- and post-treatment across melanoma subtypes including cutaneous, acral, mucosal, and uveal melanoma. Across subtypes, 33 oxylipin metabolites were analyzed, with limited variation observed overall. Prostaglandin J2 was more abundant in rare melanoma subtypes including acral, mucosal, and uveal melanoma compared to cutaneous melanoma. Conclusions: Despite limited variation of serum oxylipin molecular species by subtype and response status, we observed significant differences in Prostaglandin J2 which could serve as a potential biomarker for immune checkpoint inhibitor therapy response in melanoma. However, further investigation is warranted to explore the role of oxylipins in immune response modulation. ### Competing Interest Statement The authors have declared no competing interest.

BRAF kinase fusions are a form of structural variation in the genome and are recurrent events in driver-negative melanomas. While BRAF fusions reproducibly conserve the kinase domain, there is genetic variability with 5' gene partners and specific BRAF breakpoints. We investigated how genetic diversity of BRAF kinase fusions affects dimeric signaling and ERK activation. We overexpressed BRAF fusions with 5' gene partners including AGK, ZKSCAN1, ARMC10, PPFIBP2, and TRIM24 and found fusion dependent signaling and inhibitor sensitivity. Despite the development of next generation RAF inhibitors, there was paradoxical ERK phosphorylation with multiple pan-RAF inhibitors, which was ameliorated in certain BRAF fusions with vertical RAF/MEK inhibition using trametinib and LY3009120. Collectively, we observed some fusion-dependent effects but also tumor growth suppression and resolution of paradoxical activation with vertical pathway inhibition. ### Competing Interest Statement The authors have declared no competing interest.

Pancreatic ductal adenocarcinoma (PDAC) presents at advanced stages and is refractory to most treatment modalities. Wnt signaling activation plays a critical role in proliferation and chemotherapeutic resistance. Minimal media conditions, growth factor dependency, and Wnt dependency were determined via Wnt inhibition for seven patient derived organoids (PDOs) derived from pancreatic tumor organoid libraries (PTOL). Organoids demonstrating response in vitro were assessed in vivo using patient-derived xenografts. Wnt (in)dependent gene signatures were identified for each organoid. Panc269 demonstrated a trend of reduced organoid growth when treated with ETC-159 in combination with paclitaxel or gemcitabine as compared with chemotherapy or ETC-159 alone. Panc320 demonstrated a more pronounced anti-proliferative effect in the combination of ETC-159 and paclitaxel but not with gemcitabine. Panc269 and Panc320 were implanted into nude mice and treated with ETC-159, paclitaxel, and gemcitabine as single agents and in combination. The combination of ETC-159 and paclitaxel demonstrated an anti-tumor effect greater than ETC-159 alone. Extent of combinatory treatment effect were observed to a lesser extent in the Panc320 xenograft. Wnt (in)dependent gene signatures of Panc269 and 320 were consistent with the phenotypes displayed. Gene expression of several key Wnt genes assessed via RT-PCR demonstrated notable fold change following treatment in vivo. Each pancreatic organoid demonstrated varied niche factor dependencies, providing an avenue for targeted therapy, supported through growth analysis following combinatory treatment of Wnt inhibitor and standard chemotherapy in vitro. The clinical utilization of this combinatory treatment modality in pancreatic cancer PDOs has thus far been supported in our patient-derived xenograft models treated with Wnt inhibitor plus paclitaxel or gemcitabine. Gene expression analysis suggests there are key Wnt genes that contribute to the Wnt (in)dependent phenotypes of pancreatic tumors, providing plausible mechanistic explanation for Wnt (in)dependency and susceptibility or resistance to treatment on the genotypic level.

Background Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with a poor prognosis. Doxorubicin is part of standard curative therapy for TNBC, but chemotherapy resistance remains an important clinical challenge. Bocodepsin (OKI-179) is a small molecule class I histone deacetylase (HDAC) inhibitor that promotes apoptosis in TNBC preclinical models. The purpose of this study was to investigate the combination of bocodepsin and doxorubicin in preclinical TNBC models and evaluate the impact on terminal cell fate, including apoptosis and senescence. Methods TNBC cell lines were treated with doxorubicin and CellTiter-Glo was used to assess proliferation and determine doxorubicin sensitivity. Select cell lines were treated with OKI-005 (in vitro version of bocodepsin) and doxorubicin and assessed for proliferation, apoptosis as measured by Annexin V/PI, and cell cycle by flow cytometry. Immunoblotting was used to assess changes in mediators of apoptosis, cell cycle arrest, and senescence. Senescence was measured by the senescence-associated β-galactosidase assay. An MDA-MB-231 xenograft in vivo model was treated with bocodepsin, doxorubicin, or the combination and assessed for inhibition of tumor growth. shRNA knockdown of p53 was performed in the CAL-51 cell line and proliferation, apoptosis and senescence were assessed in response to combination treatment. Results OKI-005 and doxorubicin resulted in synergistic antiproliferative activity in TNBC cells lines regardless of p53 mutation status. The combination led to increased apoptosis and decreased senescence. In vivo, the combination resulted in increased tumor growth inhibition compared to either single agent. shRNA knock-down of p53 led to increased doxorubicin-induced senescence that was decreased with the addition of OKI-005 in vitro. Conclusion The addition of bocodepsin to doxorubicin resulted in synergistic antiproliferative activity in vitro, improved tumor growth inhibition in vivo, and promotion of apoptosis which makes this a promising combination to overcome doxorubicin resistance in TNBC. Bocodepsin is currently in clinical development and has a favorable toxicity profile compared to other HDAC inhibitors supporting the feasibility of evaluating this combination in patients with TNBC.

Despite advancements in treating cutaneous melanoma, patients with acral and mucosal (A/M) melanomas still have limited therapeutic options and poor prognoses. We analyzed 156 melanomas (101 cutaneous, 28 acral, and 27 mucosal) using the Foundation One cancer-gene specific clinical testing platform and identified new, potentially targetable genomic alterations (GAs) in specific anatomic sites of A/M melanomas. Using novel pre-clinical models of A/M melanoma, we demonstrate that several GAs and corresponding oncogenic pathways associated with cutaneous melanomas are similarly targetable in A/M melanomas. Other alterations, including MYC and CRKL amplifications, were unique to A/M melanomas and susceptible to indirect targeting using the BRD4 inhibitor JQ1 or Src/ABL inhibitor dasatinib, respectively. We further identified new, actionable A/M-specific alterations, including an inactivating NF2 fusion in a mucosal melanoma responsive to dasatinib in vivo. Our study highlights new molecular differences between cutaneous and A/M melanomas, and across different anatomic sites within A/M, which may change clinical testing and treatment paradigms for these rare melanomas.

Lysophosphatidic acid (LPA) is a bioactive lipid which increases in concentration locally and systemically across different cancer types. Yet, the exact mechanism(s) of how LPA affects CD8 T cell immunosurveillance during tumor progression remain unknown. We show LPA receptor (LPAR) signaling by CD8 T cells promotes tolerogenic states via metabolic reprogramming and potentiating exhaustive-like differentiation to modulate anti-tumor immunity. We found LPA levels predict response to immunotherapy and Lpar5 signaling promotes cellular states associated with exhausted phenotypes on CD8 T cells. Importantly, we show that Lpar5 regulates CD8 T cell respiration, proton leak, and reactive oxygen species. Together, our findings reveal that LPA serves as a lipid-regulated immune checkpoint by modulating metabolic efficiency through LPAR5 signaling on CD8 T cells. Our study offers key insights into the mechanisms governing adaptive anti-tumor immunity and demonstrates LPA could be exploited as a T cell directed therapy to improve dysfunctional anti-tumor immunity.

The prevalence of atopic diseases has increased with atopic dermatitis (AD) as the earliest manifestation. We assessed if molecular risk factors in atopic mothers influence their infants' susceptibility to an atopic disease.

Die Prävalenz atopischer Erkrankungen nimmt stetig zu, wobei die atopische Dermatitis (AD) die früheste Erscheinungsform darstellt. Bei deren Entstehung spielen genetische Faktoren, Umweltnoxen und in utero Expositionen eine wichtige Rolle. Da bisher weitestgehend unbekannt ist, welche molekularen Mechanismen an der Übertragung atopischer Erkrankungen von der Mutter auf das Kind beteiligt sein könnten, wurden 300 schwangere Frauen mit oder ohne atopischem Phänotyp rekrutiert und ihre Neugeborenen in eine prospektive Geburtskohorte eingeschlossen.

Immune checkpoint inhibitors have been found to be effective in metastatic MSI-high colorectal cancers (CRC), however, have no efficacy in microsatellite stable (MSS) cancers, which comprise the majority of mCRC cases. Cabozantinib is a small molecule multi-tyrosine kinase inhibitor that is FDA approved in advanced renal cell, medullary thyroid, and hepatocellular carcinoma. Using Human Immune System (HIS) mice, we tested the ability of cabozantinib to prime MSS-CRC tumors to enhance the potency of immune checkpoint inhibitor nivolumab. In four independent experiments, we implanted distinct MSS-CRC patient-derived xenografts (PDXs) into the flanks of humanized BALB/c-Rag2nullIl2rγnullSirpαNOD (BRGS) mice that had been engrafted with human hematopoietic stem cells at birth. For each PDX, HIS-mice cohorts were treated with vehicle, nivolumab, cabozantinib, or the combination. In three out of the four models, the combination had a lower tumor growth rate compared to vehicle or nivolumab-treated groups. Furthermore, interrogation of the HIS in immune organs and tumors by flow cytometry revealed increased Granzyme B+, TNFα+ and IFNγ+ CD4+ T cells among the human tumor infiltrating leukocytes (TIL) that correlated with reduced tumor growth in the combination-treated HIS-mice. Notably, slower growth correlated with increased expression of the CD4+ T cell ligand, HLA-DR, on the tumor cells themselves. Finally, the cabozantinib/nivolumab combination was tested in comparison to cobimetinib/atezolizumab. Although both combinations showed tumor growth inhibition, cabozantinib/nivolumab had enhanced cytotoxic IFNγ and TNFα+ T cells. This pre-clinical in vivo data warrants testing the combination in clinical trials for patients with MSS-CRC.

Despite adequate infection prophylaxis, variation in self-reported quality of life (QOL) throughout the intravenous immunoglobulin (IVIG) infusion cycle is a widely reported but infrequently studied phenomenon. To better understand this phenomenon, subjects with humoral immunodeficiency receiving replacement doses of IVIG were studied over 3 infusion cycles. Questionnaire data from 6 time points spread over 3 IVIG infusions cycles (infusion day and 7 days after each infusion) were collected in conjunction with monitoring the blood for number of regulatory T-cells (Treg) and levels of 40 secreted analytes: primarily cytokines, chemokines, and growth factors. At day 7, self-reported well-being increased, and self-reported fatigue decreased, reflecting an overall improvement in QOL 7 days after infusion. Over the same period, percentage of Treg cells in the blood increased (p<0.01). Multiple inflammatory chemokine and cytokine levels increased in the blood by 1 hour after infusion (CCL4 (MIP-1b), CCL3 (MIP-1a), CCL2 (MCP-1), TNF-α, granzyme B, IL-10, IL-1RA, IL-8, IL-6, GM-CSF, and IFN- γ). The largest changes in analytes occurred in subjects initiated on IVIG during the study. A significant decrease in IL-25 (IL-17E) following infusion was seen in most intervals among subjects already receiving regular infusions prior to study entry. These findings reveal several short-term effects of IVIG given in replacement doses to patients with humoral immunodeficiency: QOL consistently improves in the first week of infusion, levels of a collection of monocyte-associated cytokines increase immediately after infusion whereas IL-25 levels decrease, and Treg levels increase. Moreover, patients that are new to IVIG experience more significant fluctuations in cytokine levels than those receiving it regularly.

Cutaneous melanoma (CM) has one of the highest response rates across cancers to immune checkpoint blockade (ICB) therapies, but mucosal melanoma (MM) responds poorly to ICB. MM patients have very few effective treatments options, making it critical to understand and improve anti-tumor immunity in these patients. MM is a rare subtype of melanoma, accounting for approximately 1% of all malignant melanoma diagnoses. MM occurs on mucosal surfaces such as nasal, vaginal, and anorectal, which provide a highly unique tumor microenvironment compared to CM arising in the skin. We compared the tumor immune microenvironment between CM and MM by multiplex immunofluorescence and discovered MM has decreased frequency of infiltrating immune cells. Using RNA sequencing, we investigated intra-tumoral differences in global gene expression between CM and MM that might explain the lack of immune cell infiltration and ICB response in MM. Compared to CM, MM had decreased expression of numerous innate immune genes in the RIG-I pathogen-sensing pathway. The RIG-I pathway produces an inflammatory response to destroy foreign pathogens, but is also important for ICB sensitivity and anti-tumor immunity in CM. Studies in CM demonstrate the RIG-I pathway enhances antigen presentation and promotes a favorable immune cell profile in the tumor microenvironment. Interestingly, the tumor microbiome, which is vastly different between CM and MM, has been shown to regulate the RIG-I pathway. We measured total fungal and bacterial load in MM and CM primary tumors and found increased levels of both bacteria and fungi in MM compared to CM. This raises the possibility that microbiome-mediated suppression of the RIG-I pathway underlies poor anti-tumor immunity and ICB response in MM, and that re-activation of this pathway may be a novel therapeutic strategy for overcoming ICB resistance in MM patients. Direct RIG-I agonists are still in clinical development, but FDA-approved hypomethylating agents, particularly 5’ aza-deoxycytidine (decitabine), can activate the RIG-I pathway and improve anti-tumor immunity by increasing the expression of RIG-I pathway activating genes, endogenous RNA retroviruses, tumor antigens, and natural killer (NK) cell ligands. We treated MM cell lines with decitabine and observed strong induction of RIG-I pathway genes, NK ligands, and tumor antigens. In conclusion, we have identified the loss of intra-tumoral RIG-I signaling as a potential microbiome-mediated mechanism underlying the poor tumor immune microenvironment and ICB response in MM. Combining decitabine, which is FDA-approved and now available orally, with ICB represents a novel treatment strategy for these difficult to treat MM patients that can be rapidly translated into the clinic. Citation Format: Morgan MacBeth, Richard Tobin, Robert Van Gulick, Martin D. McCarter, William A. Robinson, Kasey L. Couts. Loss of intra-tumoral RIG-I immune signaling is a potential microbiome-mediated mechanism underlying poor anti-tumor immunity and immunotherapy resistance in mucosal melanoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO048.

e21593 Background: Patients with cutaneous (CM) or acral (AM) melanoma have high response rates to immune checkpoint blockade (ICB) therapies including anti-CTLA4, anti-PD1, and their combination. In contrast, patients with mucosal (MM) or uveal (UM) melanoma have low ICB response rates and a poorer overall prognosis. In this study, we analyzed melanoma transcriptomes to identify potential mechanisms underlying ICB resistance in MM and UM. We also evaluated epigenetic modifying drugs as potential agents to improve ICB responses in these patients. Methods: RNA sequencing was performed on 13 MM, 8 AM, and 24 CM patient-derived xenograft tumors. These data were analyzed along with published RNA sequencing data for 80 UM and 443 CM tumors. MM and UM cells were treated with several different classes of epigenetic modifying drugs, and the ICB-resistant YUMM1.7 mouse melanoma syngeneic model was used to evaluate the tumor growth effect of one particular drug, decitabine, at low (2.5 ug) and intermediate (10 ug) doses in combination with anti-PD1 (200 ug). Results: We identified 2513 differentially expressed genes (DEG) between MM and CM tumors (ANODEV adjusted p-value < 0.05, FDR < 0.1) compared to only 495 DEG between AM and CM. Ingenuity Pathway Analysis (IPA) analysis of the 2513 DEG in MM identified the RIG-I-like receptor (RLR) pathway as inhibited in MM versus CM ( p < 1x10 -5 ). The RLR pathway was also inhibited in ICB-resistant CM versus ICI-sensitive (anti-CTLA4, p < 0.006 and anti-PD1, p < 0.0002) and in UM versus CM ( p < 1x10 -8 ), but was not inhibited in AM. The RLR pathway is an innate immune pathogen sensing pathway that is critical for ICB responses in CM; therefore, we sought to identify FDA-approved drugs which activate the RLR pathway in MM and UM. Since RLR signaling is frequently repressed by epigenetic silencing, we screened direct DNMT1 inhibitors 5’-azacitidine (AZA) and 5’-aza-deoxycitidine (decitabine, DEC), several indirect DNMT1 inhibitors, and two HDAC inhibitors. We found that only DEC strongly induced the expression of RLR pathway genes in MM, UM, and ICB-resistant CM cells. Compared to vehicle, treatment of YUMM1.7 tumors with single agent anti-PD1 ( p > 0.87) and low dose ( p > 0.90) or intermediate dose ( p > 0.80) DEC had no effect on tumor growth. However, the addition of either low or intermediate dose DEC to anti-PD1 showed a trend of decreased tumor growth (low dose, 31% decrease, p = 0.22 and intermediate dose, 41% decrease, p = 0.55). Conclusions: We identified low RLR pathway signaling as one possible mechanism underlying ICB resistance in MM and UM. We show that an FDA-approved DNMT1 inhibitor, decitabine, strongly induces RLR pathway genes in MM and UM cells in vitro and leads to tumor growth suppression in combination with anti-PD1 in vivo. Therefore, co-administration of decitabine and anti-PD1 agents is a rational and novel strategy for improving outcomes for MM and UM patients.

Although treatment options for melanoma patients have expanded in recent years with the approval of immunotherapy and targeted therapy, there is still an unmet need for new treatment options for patients that are ineligible for, or resistant to these therapies. BH3 mimetics, drugs that mimic the activity of pro-apoptotic BCL2 family proteins, have recently achieved remarkable success in the clinical setting. The combination of BH3 mimetic ABT-199 (venetoclax) plus azacitidine has shown substantial benefit in treating acute myelogenous leukemia. We evaluated the efficacy of various combinations of BH3 mimetic + azacitidine in fourteen human melanoma cell lines from cutaneous, mucosal, acral and uveal subtypes. Using a combination of cell viability assay, BCL2 family knockdown cell lines, live cell imaging, and sphere formation assay, we found that combining inhibition of MCL1, an anti-apoptotic BCL2 protein, with azacitidine had substantial pro-apoptotic effects in multiple melanoma cell lines. Specifically, this combination reduced cell viability, proliferation, sphere formation, and induced apoptosis. In addition, this combination is highly effective at reducing cell viability in rare mucosal and uveal subtypes. Overall, our data suggest this combination as a promising therapeutic option for some patients with melanoma and should be further explored in clinical trials.

Current treatment for patients with metastatic melanoma include molecular-targeted therapies and immune checkpoint inhibitors. However, a subset of melanomas are difficult-to-treat. These melanomas include those without the genetic markers for targeted therapy, non-responsive to immunotherapy, and those who have relapsed or exhausted their therapeutic options. Therefore, it is necessary to understand and explore other biological processes that may provide new therapeutic approaches. One of most appealing is targeting the apoptotic/anti-apoptotic system that is effective against leukemia. We used genetic knockdown and pharmacologic approaches of BH3 mimetics to target anti-apoptotic BCL2 family members and identified MCL1 and BCLXL as crucial pro-survival members in melanoma. We then examined the effects of combining BH3 mimetics to target MCL1 and BCLXL in vitro and in vivo. These include clinical-trial-ready compounds such as ABT-263 (Navitoclax) and S63845/S64315 (MIK655). We used cell lines derived from patients with difficult-to-treat melanomas. In vitro, the combined inhibition of MCL1 and BCLXL resulted in significantly effective cell killing compared to single-agent treatment (p < 0.05) in multiple assays, including sphere assays. The combination-induced cell death was independent of BIM, and NOXA. Recapitulated in our mouse xenograft model, the combination inhibited tumor growth, reduced sphere-forming capacity (p < 0.01 and 0.05, respectively), and had tolerable toxicity (p > 0.40). Taken together, this study suggests that dual targeting of MCL1 and BCLXL should be considered as a treatment option for difficult-to-treat melanoma patients.

Subungual melanomas (SUM) arise beneath the nails of the hands and feet, and account for 0.7-3.5% of all malignant melanomas. Most studies include SUM in the category of acral melanoma, but understanding the specific features of SUM is critical for improving patient care. In this study, we performed a site-specific comparison of the clinical and molecular features between 54 cases of SUM and 78 cases of nonsubungual acral melanoma. Compared to patients with acral melanoma, patients with SUM were younger at diagnosis, had a higher prevalence of primary melanomas on the hand, and had more frequent reports of previous trauma at the tumor site. SUM was deeper than acral melanoma at diagnosis, which correlated with an increased frequency of metastases. Analysis of common melanoma driver genes revealed KIT and KRAS mutations were predominantly found in SUM, whereas BRAF and NRAS mutations occurred almost exclusively in acral melanoma. We also discovered molecular differences in the cell cycle pathway, where CDK4/CCND1 amplifications were more frequent in SUM and CDKN2A/B loss occurred mostly in acral melanoma, and in the PI3K/mTOR pathway, where RICTOR amplification and TSC1 K587R mutations were exclusively in SUM and PTEN loss and AKT1 mutations were exclusively in acral melanoma. Comparison of hand versus foot tumors revealed more frequent ulceration of SUM foot tumors, which correlated with more distal metastases and poorer overall survival. In summary, we find SUM are both clinically and molecularly distinct from acral melanoma, and our data suggest KIT, CDK4/6, and mTOR inhibitors may be particularly relevant and effective treatments for patients with SUM.

Abstract Treatment options for melanoma have progressed in recent years, with the rise of targeted therapy and immunotherapy. Options are still limited for patients that are BRAF wildtype, treatment refractory, and patients with non-sun exposed melanoma subtypes that typically do not respond to standard therapies. Therefore, it is necessary to explore other biological processes that may provide new therapeutic approaches. One of most appealing is the BCL-2 family of apoptotic/anti-apoptotic system that is effective in leukemias, with BH3 mimetics venetoclax alone or in combination with the hypomethylating agent 5-azacitidine. We examined similar combinations on the group of eleven patient-derived and commercially available melanoma cell lines. The BH3 mimetics we tested include MCL1 inhibitors (S63845, AMG 176, AZD5991), an inhibitor of BCL2, BCLXL, and BCLW (navitoclax/ABT-263), a BCL2 inhibitor (venotoclax/ABT-199), and a BCLXL inhibitor (A1331852). Our data showed that treatments with MCL1 inhibitors plus 5-azacitidine are effective in reducing melanoma cell viability. S63845 was the most potent compound when combined with 5-azacitidine, with nine out of eleven (~80%) melanoma cell lines achieving less than 50% viability at the highest dose. There was an overall weaker response to treatment with 5-azacitidine in combination with ABT-263 or A1331852, with only 54% and 45% of cell lines responding at the highest dose, respectively. Response to ABT-199 with 5-Azacitidine was highly limited, 18% of cell lines responded at the highest concentration. Our data demonstrate that treatment with S63845 in combination with 5-azacitidine is highly effective in cell lines from a variety of melanoma subtypes, including the rare uveal and mucosal subtypes. In vivo and mechanistic experiments are planned. The novel combination of the MCL1 inhibitor S63845 and 5-azacitidine may be a promising alternative therapeutic target in melanoma. Citation Format: Chiara Dart, Nabanita Mukherjee, Carol Amato, Robert J. Van Gulick, Morgan MacBeth, Kasey Couts, Jacqueline Turner, Stacey Bagby, Mayumi Fujita, William Robinson, Yiqun Shellman. The therapeutic potential of 5-Azacitidine plus various BH3 mimetics in melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1805.

The tumor suppressor TP53 is the most frequently mutated gene product in human cancer. Close to half of all solid tumors carry inactivating mutations in the TP53 gene, while in the remaining cases, TP53 activity is abrogated by other oncogenic events, such as hyperactivation of its endogenous repressors MDM2 or MDM4. Despite identification of hundreds of genes regulated by this transcription factor, it remains unclear which direct target genes and downstream pathways are essential for the tumor suppressive function of TP53. We set out to address this problem by generating multiple genomic data sets for three different cancer cell lines, allowing the identification of distinct sets of TP53-regulated genes, from early transcriptional targets through to late targets controlled at the translational level. We found that although TP53 elicits vastly divergent signaling cascades across cell lines, it directly activates a core transcriptional program of ∼100 genes with diverse biological functions, regardless of cell type or cellular response to TP53 activation. This core program is associated with high-occupancy TP53 enhancers, high levels of paused RNA polymerases, and accessible chromatin. Interestingly, two different shRNA screens failed to identify a single TP53 target gene required for the anti-proliferative effects of TP53 during pharmacological activation in vitro. Furthermore, bioinformatics analysis of thousands of cancer genomes revealed that none of these core target genes are frequently inactivated in tumors expressing wild-type TP53. These results support the hypothesis that TP53 activates a genetically robust transcriptional program with highly distributed tumor suppressive functions acting in diverse cellular contexts.