Differential histone acetylation and super-enhancer regulation underlie melanoma cell dedifferentiation.

Dedifferentiation or phenotype switching refers to the transition from a proliferative to an invasive cellular state. We previously identified a 122-gene epigenetic gene signature that classifies primary melanomas as low versus high risk (denoted as Epgn1 or Epgn3). We found that the transcriptomes of the Epgn1 low-risk and Epgn3 high-risk cells are similar to the proliferative and invasive cellular states, respectively. These signatures were further validated in melanoma tumor samples. Examination of the chromatin landscape revealed differential H3K27 acetylation in the Epgn1 low-risk versus Epgn3 high-risk cell lines that corroborated with a differential super-enhancer and enhancer landscape. Melanocytic lineage genes (MITF, its targets and regulators) were associated with super-enhancers in the Epgn1 low-risk state, whereas invasiveness genes were linked with Epgn3 high-risk status. We identified the ITGA3 gene as marked by a super-enhancer element in the Epgn3 invasive cells. Silencing of ITGA3 enhanced invasiveness in both in vitro and in vivo systems, suggesting it as a negative regulator of invasion. In conclusion, we define chromatin landscape changes associated with Epgn1/Epgn3 and phenotype switching during early steps of melanoma progression that regulate transcriptional reprogramming. This super-enhancer and enhancer-driven epigenetic regulatory mechanism resulting in major changes in the transcriptome could be important in future therapeutic targeting efforts.

Molecular mechanisms in colitis-associated colorectal cancer.

Sustained chronic inflammation of the large intestine leads to tissue damage and repair, which is associated with an increased incidence of colitis-associated colorectal cancer (CAC). The genetic makeup of CAC is somewhat similar to sporadic colorectal carcinoma (sCRC), but there are differences in the sequence and timing of alterations in the carcinogenesis process. Several models have been developed to explain the development of CAC, particularly the "field cancerization" model, which proposes that chronic inflammation accelerates mutagenesis and selects for the clonal expansion of phenotypically normal, pro-tumorigenic cells. In contrast, the "Big Bang" model posits that tumorigenic clones with multiple driver gene mutations emerge spontaneously. The details of CAC tumorigenesis-and how they differ from sCRC-are not yet fully understood. In this Review, we discuss recent genetic, epigenetic, and environmental findings related to CAC pathogenesis in the past five years, with a focus on unbiased, high-resolution genetic profiling of non-dysplastic field cancerization in the context of inflammatory bowel disease (IBD).

Etiology of super-enhancer reprogramming and activation in cancer.

Super-enhancers are large, densely concentrated swaths of enhancers that regulate genes critical for cell identity. Tumorigenesis is accompanied by changes in the super-enhancer landscape. These aberrant super-enhancers commonly form to activate proto-oncogenes, or other genes upon which cancer cells depend, that initiate tumorigenesis, promote tumor proliferation, and increase the fitness of cancer cells to survive in the tumor microenvironment. These include well-recognized master regulators of proliferation in the setting of cancer, such as the transcription factor MYC which is under the control of numerous super-enhancers gained in cancer compared to normal tissues. This Review will cover the expanding cell-intrinsic and cell-extrinsic etiology of these super-enhancer changes in cancer, including somatic mutations, copy number variation, fusion events, extrachromosomal DNA, and 3D chromatin architecture, as well as those activated by inflammation, extra-cellular signaling, and the tumor microenvironment.

AKT activation because of PTEN loss upregulates xCT via GSK3ß/NRF2, leading to inhibition of ferroptosis in PTEN-mutant tumor cells.

Here, we show that the tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) sensitizes cells to ferroptosis, an iron-dependent form of cell death, by restraining the expression and activity of the cystine/glutamate antiporter system Xc- (xCT). Loss of PTEN activates AKT kinase to inhibit GSK3ß, increasing NF-E2 p45-related factor 2 (NRF2) along with transcription of one of its known target genes encoding xCT. Elevated xCT in Pten-null mouse embryonic fibroblasts increases the flux of cystine transport and synthesis of glutathione, which enhances the steady-state levels of these metabolites. A pan-cancer analysis finds that loss of PTEN shows evidence of increased xCT, and PTEN-mutant cells are resistant to ferroptosis as a consequence of elevated xCT. These findings suggest that selection of PTEN mutation during tumor development may be due to its ability to confer resistance to ferroptosis in the setting of metabolic and oxidative stress that occurs during tumor initiation and progression.

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in ß-catenin activated melanomas.

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/ß-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the 'cold tumor' phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by ß-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).

Novel Allosteric Inhibitor-Derived AKT Proteolysis Targeting Chimeras (PROTACs) Enable Potent and Selective AKT Degradation in KRAS/BRAF Mutant Cells.

AKT is an important target for cancer therapeutics. Significant advancements have been made in developing ATP-competitive and allosteric AKT inhibitors. Recently, several AKT proteolysis targeting chimeras (PROTACs) derived from ATP-competitive AKT inhibitors have been reported, including MS21. While MS21 potently degraded AKT and inhibited the growth in tumor cells harboring PI3K/PTEN pathway mutation, it was largely ineffective in degrading AKT in KRAS/BRAF mutated cells as a single agent. To overcome the AKT degradation resistance in KRAS/BRAF mutated cells, we developed novel AKT PROTACs derived from an AKT allosteric inhibitor, including degrader 62 (MS15). 62 displayed potent and selective AKT degradation activity and potent antiproliferative activity in KRAS/BRAF mutated cancer cells, in addition to PI3K/PTEN mutated cancer cells. Furthermore, 62 was bioavailable in mice through intraperitoneal administration. Overall, 62 is a valuable chemical tool to degrade AKT in cells harboring KRAS/BRAF mutation and expands the tool box for pharmacologically modulating AKT.

A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma.

Tumors exhibit enhancer reprogramming compared to normal tissue. The etiology is largely attributed to cell-intrinsic genomic alterations. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon, we find divergent epigenetic landscapes between CRC tumors and cell lines. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over normal. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. We restore this super-enhancer and its expressed gene, PDZK1IP1, following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating cell-extrinsic etiology. We demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway. We show this activation enables efficient growth under oxidative conditions, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the significance of epigenomic profiling on primary specimens.

Inpatient Administration of Alpha-1-Adrenergic Receptor Blocking Agents Reduces Mortality in Male COVID-19 Patients.

Apha-1-adrenergic receptor antagonists (α1-blockers) can suppress pro-inflammatory cytokines, thereby potentially improving outcomes among patients with COVID-19. Accordingly, we evaluated the association between α1-blocker exposure (before or during hospitalization) and COVID-19 in-hospital mortality. We identified 2,627 men aged 45 or older who were admitted to Mount Sinai hospitals with COVID-19 between February 24 and May 31, 2020, in New York. Men exposed to α1-blockers (N = 436) were older (median age 73 vs. 64 years, P < 0.001) and more likely to have comorbidities than unexposed men (N = 2,191). Overall, 777 (29.6%) patients died in hospital, and 1,850 (70.4%) were discharged. Notably, we found that α1-blocker exposure was independently associated with improved in-hospital mortality in a multivariable logistic analysis (OR 0.699; 95% CI, 0.498-0.982; P = 0.039) after adjusting for patient demographics, comorbidities, and baseline vitals and labs. The protective effect of α1-blockers was stronger among patients with documented inpatient exposure to α1-blockers (OR 0.624; 95% CI 0.431-0.903; P = 0.012). Finally, age-stratified analyses suggested variable benefit from inpatient α1-blocker across age groups: Age 45-65 OR 0.483, 95% CI 0.216-1.081 (P = 0.077); Age 55-75 OR 0.535, 95% CI 0.323-0.885 (P = 0.015); Age 65-89 OR 0.727, 95% CI 0.484-1.092 (P = 0.124). Taken together, clinical trials to assess the therapeutic value of α1-blockers for COVID-19 complications are warranted.

Discovery of Potent, Selective, and In Vivo Efficacious AKT Kinase Protein Degraders via Structure-Activity Relationship Studies.

We recently reported a potent, selective, and in vivo efficacious AKT degrader, MS21, which is a von Hippel-Lindau (VHL)-recruiting proteolysis targeting chimera (PROTAC) based on the AKT inhibitor AZD5363. However, no structure-activity relationship (SAR) studies that resulted in this discovery have been reported. Herein, we present our SAR studies that led to the discovery of MS21, another VHL-recruiting AKT degrader, MS143 (compound 20) with similar potency as MS21, and a novel cereblon (CRBN)-recruiting PROTAC, MS5033 (compound 35). Compounds 20 and 35 induced rapid and robust AKT degradation in a concentration- and time-dependent manner via hijacking the ubiquitin-proteasome system. Compound 20 suppressed cell growth more effectively than AZD5363 in multiple cancer cell lines. Furthermore, 20 and 35 displayed good plasma exposure levels in mice and are suitable for in vivo efficacy studies. Lastly, compound 20 effectively suppressed tumor growth in vivo in a xenograft model without apparent toxicity.

Design, Synthesis, and Evaluation of Potent, Selective, and Bioavailable AKT Kinase Degraders.

The serine/threonine kinase AKT functions as a critical node of the phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (m-TOR) signaling pathway. Aberrant activation and overexpression of AKT are strongly correlated with numerous human cancers. To date, only two AKT degraders with no structure-activity relationship (SAR) results have been reported. Through extensive SAR studies on various linkers, E3 ligase ligands, and AKT binding moieties, we identified two novel and potent AKT proteolysis targeting chimera (PROTAC) degraders: von Hippel-Lindau (VHL)-recruiting degrader 13 (MS98) and cereblon (CRBN)-recruiting degrader 25 (MS170). These two compounds selectively induced robust AKT protein degradation, inhibited downstream signaling, and suppressed cancer cell proliferation. Moreover, these two degraders exhibited good plasma exposure levels in mice through intraperitoneal injection. Overall, our comprehensive SAR studies led to the discovery of degraders 13 and 25, which are potentially useful chemical tools to investigate biological and pathogenic functions of AKT in vitro and in vivo.

Cooperation Between Distinct Cancer Driver Genes Underlies Intertumor Heterogeneity in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: The pattern of genetic alterations in cancer driver genes in patients with hepatocellular carcinoma (HCC) is highly diverse, which partially explains the low efficacy of available therapies. In spite of this, the existing mouse models only recapitulate a small portion of HCC inter-tumor heterogeneity, limiting the understanding of the disease and the nomination of personalized therapies. Here, we aimed at establishing a novel collection of HCC mouse models that captured human HCC diversity. METHODS: By performing hydrodynamic tail-vein injections, we tested the impact of altering a well-established HCC oncogene (either MYC or ß-catenin) in combination with an additional alteration in one of eleven other genes frequently mutated in HCC. Of the 23 unique pairs of genetic alterations that we interrogated, 9 were able to induce HCC. The established HCC mouse models were characterized at histopathological, immune, and transcriptomic level to identify the unique features of each model. Murine HCC cell lines were generated from each tumor model, characterized transcriptionally, and used to identify specific therapies that were validated in vivo. RESULTS: Cooperation between pairs of driver genes produced HCCs with diverse histopathology, immune microenvironments, transcriptomes, and drug responses. Interestingly, MYC expression levels strongly influenced ß-catenin activity, indicating that inter-tumor heterogeneity emerges not only from specific combinations of genetic alterations but also from the acquisition of expression-dependent phenotypes. CONCLUSIONS: This novel collection of murine HCC models and corresponding cell lines establishes the role of driver genes in diverse contexts and enables mechanistic and translational studies.

PTEN interacts with the transcription machinery on chromatin and regulates RNA polymerase II-mediated transcription.

Regulation of RNA polymerase II (RNAPII)-mediated transcription controls cellular phenotypes such as cancer. Phosphatase and tensin homologue deleted on chromosome ten (PTEN), one of the most commonly altered tumor suppressors in cancer, affects transcription via its role in antagonizing the PI3K/AKT signaling pathway. Using co-immunoprecipitations and proximal ligation assays we provide evidence that PTEN interacts with AFF4, RNAPII, CDK9, cyclin T1, XPB and CDK7. Using ChIP-seq, we show that PTEN co-localizes with RNAPII and binds to chromatin in promoter and putative enhancer regions identified by histone modifications. Furthermore, we show that loss of PTEN affects RNAPII occupancy in gene bodies and further correlates with gene expression changes. Interestingly, PTEN binds to promoters and negatively regulates the expression of genes involved in transcription including AFF4 and POL2RA, which encodes a subunit of RNAPII. Loss of PTEN also increased cells' sensitivity to transcription inhibition via small molecules, which could provide a strategy to target PTEN-deficient cancers. Overall, our work describes a previously unappreciated role of nuclear PTEN, which by interacting with the transcription machinery in the context of chromatin exerts an additional layer of regulatory control on RNAPII-mediated transcription.

Molecular Pathways: Targeting the PI3K Pathway in Cancer-BET Inhibitors to the Rescue.

The PI3K signaling pathway is a complex and tightly regulated network that is critical for many physiologic processes, such as cell growth, proliferation, metabolism, and survival. Aberrant activation of this pathway can occur through mutation of almost any of its major nodes and has been implicated in a number of human diseases, including cancer. The high frequency of mutations in this pathway in multiple types of cancer has led to the development of small-molecule inhibitors of PI3K, several of which are currently in clinical trials. However, several feedback mechanisms either within the PI3K pathway or in compensatory pathways can render tumor cells resistant to therapy. Recently, targeting proteins of the bromodomain and extraterminal (BET) family of epigenetic readers of histone acetylation has been shown to effectively block adaptive signaling response of cancer cells to inhibitors of the PI3K pathway, which at least in some cases can restore sensitivity. BET inhibitors also enforce blockade of the MAPK, JAK/STAT, and ER pathways, suggesting they may be a rational combinatorial partner for divergent oncogenic signals that are subject to homeostatic regulation. Here, we review the PI3K pathway as a target for cancer therapy and discuss the potential use of BET inhibition to enhance the clinical efficacy of PI3K inhibitors. Clin Cancer Res; 22(11); 2605-10. ©2016 AACR.

Metformin and erlotinib synergize to inhibit basal breast cancer.

Basal-like breast cancers (BBCs) are enriched for increased EGFR expression and decreased expression of PTEN. We found that treatment with metformin and erlotinib synergistically induced apoptosis in a subset of BBC cell lines. The drug combination led to enhanced reduction of EGFR, AKT, S6 and 4EBP1 phosphorylation, as well as prevented colony formation and inhibited mammosphere outgrowth. Our data with other compounds suggested that biguanides combined with EGFR inhibitors have the potential to outperform other targeted drug combinations and could be employed in other breast cancer subtypes, as well as other tumor types, with activated EGFR and PI3K signaling. Analysis of BBC cell line alterations led to the hypothesis that loss of PTEN sensitized cells to the drug combination which was confirmed using isogenic cell line models with and without PTEN expression. Combined metformin and erlotinib led to partial regression of PTEN-null and EGFR-amplified xenografted MDA-MB-468 BBC tumors with evidence of significant apoptosis, reduction of EGFR and AKT signaling, and lack of altered plasma insulin levels. Combined treatment also inhibited xenografted PTEN null HCC-70 BBC cells. Measurement of trough plasma drug levels in xenografted mice and a separately performed pharmacokinetics modeling study support possible clinical translation.

Molecular pathways: intercellular PTEN and the potential of PTEN restoration therapy.

Phosphatase and Tensin homolog deleted on chromosome Ten (PTEN) acts as a tumor suppressor through both PI3K-dependent and -independent mechanisms. Reduced PTEN activity has been shown to affect not only tumor cell proliferation and survival but also the microenvironmental context in which nascent tumors develop. As a result of the multifaceted tumor-suppressive roles of PTEN, tumors evolve by selecting for clones in which PTEN activity is lost. PTEN activity within tumors can be modulated in numerous ways, including direct mutation, epigenetic regulation, and amplification or mutation of other proteins that can regulate or degrade PTEN. These events functionally prevent PTEN protein from acting within tumor cells. Paracrine roles for PTEN gene products (exosomal PTEN and PTEN-L) have recently been identified, through which PTEN gene products produced in one cell are able to enter recipient cells and contribute to PTEN functions. In preclinical models purified PTEN-L protein was able to enter tumor xenografts and downregulate PI3K signaling as well as cause tumor cell death. Here, we review the role of PTEN as a multifaceted tumor suppressor and reflect upon the potential for PTEN restoration therapy.

PTEN function: the long and the short of it.

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a phosphatase that is frequently altered in cancer. PTEN has phosphatase-dependent and -independent roles, and genetic alterations in PTEN lead to deregulation of protein synthesis, the cell cycle, migration, growth, DNA repair, and survival signaling. PTEN localization, stability, conformation, and phosphatase activity are controlled by an array of protein-protein interactions and post-translational modifications. Thus, PTEN-interacting and -modifying proteins have profound effects on the tumor suppressive functions of PTEN. Moreover, recent studies identified mechanisms by which PTEN can exit cells, via either exosomal export or secretion, and act on neighboring cells. This review focuses on modes of PTEN protein regulation and ways in which perturbations in this regulation may lead to disease.

Alterations of EGFR, p53 and PTEN that mimic changes found in basal-like breast cancer promote transformation of human mammary epithelial cells.

Breast cancer can be classified into different molecular subtypes with varying clinical and pathological characteristics. The basal-like breast cancer subtype represents one of the most aggressive and lethal types of breast cancer, and due to poor mechanistic understanding, it lacks targeted therapy. Many basal-like breast cancer patient samples display alterations of established drivers of cancer development, including elevated expression of EGFR, p53 inactivating mutations and loss of expression of the tumor suppressor PTEN; however, their contribution to human basal-like breast cancer pathogenesis remains ill-defined. Using non-transformed human mammary epithelial cells, we set out to determine whether altering EGFR, p53 and PTEN in different combinations could contribute to basal-like breast cancer progression through transformation of cells. Altering PTEN in combination with either p53 or EGFR in contrast to any of the single alterations caused increased growth of transformed colonies in soft agar. Concomitantly modifying all three genes led to the highest rate of cellular proliferation and the greatest degree of anchorage-independent colony formation. Results from our effort to engineer a model of BBC expressing alterations of EGFR, p53 and PTEN suggest that these changes are cooperative and likely play a causal role in basal-like breast cancer pathogenesis. Consideration should be given to targeting EGFR and restoring p53 and PTEN signaling simultaneously as a strategy for treatment of this subtype of breast cancer.

Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer.

While the small GTPase Rac1 and its effectors are well-established mediators of mitogenic and motile signaling by tyrosine kinase receptors and have been implicated in breast tumorigenesis, little is known regarding the exchange factors (Rac-GEFs) that mediate ErbB receptor responses. Here, we identify the PIP(3)-Gßγ-dependent Rac-GEF P-Rex1 as an essential mediator of Rac1 activation, motility, cell growth, and tumorigenesis driven by ErbB receptors in breast cancer cells. Notably, activation of P-Rex1 in breast cancer cells requires the convergence of inputs from ErbB receptors and a Gßγ- and PI3Kγ-dependent pathway. Moreover, we identified the GPCR CXCR4 as a crucial mediator of P-Rex1/Rac1 activation in response to ErbB ligands. P-Rex1 is highly overexpressed in human breast cancers and their derived cell lines, particularly those with high ErbB2 and ER expression. In addition to the prognostic and therapeutic implications, our findings reveal an ErbB effector pathway that is crucial for breast cancer progression.