KRAS Promotes GLI2-Dependent Transcription during Pancreatic Carcinogenesis.

Aberrant activation of GLI transcription factors has been implicated in the pathogenesis of different tumor types including pancreatic ductal adenocarcinoma. However, the mechanistic link with established drivers of this disease remains in part elusive. In this study, using a new genetically engineered mouse model overexpressing constitutively active mouse form of GLI2 and a combination of genome-wide assays, we provide evidence of a novel mechanism underlying the interplay between KRAS, a major driver of pancreatic ductal adenocarcinoma development, and GLI2 to control oncogenic gene expression. These mice, also expressing KrasG12D, show significantly reduced median survival rate and accelerated tumorigenesis compared with the KrasG12D only expressing mice. Analysis of the mechanism using RNA sequencing demonstrate higher levels of GLI2 targets, particularly tumor growth-promoting genes, including Ccnd1, N-Myc, and Bcl2, in KrasG12D mutant cells. Furthermore, chromatin immunoprecipitation sequencing studies showed that in these cells KrasG12D increases the levels of trimethylation of lysine 4 of the histone 3 (H3K4me3) at the promoter of GLI2 targets without affecting significantly the levels of other major active chromatin marks. Importantly, Gli2 knockdown reduces H3K4me3 enrichment and gene expression induced by mutant Kras. In summary, we demonstrate that Gli2 plays a significant role in pancreatic carcinogenesis by acting as a downstream effector of KrasG12D to control gene expression.

Lung adenocarcinomas without driver genes converge to common adaptive strategies through diverse genetic, epigenetic, and niche construction evolutionary pathways.

Somatic evolution selects cancer cell phenotypes that maximize survival and proliferation in dynamic environments. Although cancer cells are molecularly heterogeneous, we hypothesized convergent adaptive strategies to common host selection forces can be inferred from patterns of epigenetic and genetic evolutionary selection in similar tumors. We systematically investigated gene mutations and expression changes in lung adenocarcinomas with no common driver genes (n = 313). Although 13,461 genes were mutated in at least one sample, only 376 non-synonymous mutations evidenced positive evolutionary selection with conservation of 224 genes, while 1736 and 2430 genes exhibited ≥ two-fold increased and ≥ 50% decreased expression, respectively. Mutations under positive selection are more frequent in genes with significantly altered expression suggesting they often "hardwire" pre-existing epigenetically driven adaptations. Conserved genes averaged 16-fold higher expression in normal lung tissue compared to those with selected mutations demonstrating pathways necessary for both normal cell function and optimal cancer cell fitness. The convergent LUAD phenotype exhibits loss of differentiated functions and cell-cell interactions governing tissue organization. Conservation with increased expression is found in genes associated with cell cycle, DNA repair, p53 pathway, epigenetic modifiers, and glucose metabolism. No canonical driver gene pathways exhibit strong positive selection, but extensive down-regulation of membrane ion channels suggests decreased transmembrane potential may generate persistent proliferative signals. NCD LUADs perform niche construction generating a stiff, immunosuppressive microenvironment through selection of specific collagens and proteases. NCD LUADs evolve to a convergent phenotype through a network of interconnected genetic, epigenetic, and ecological pathways.

Molecular Characterization and Therapeutic Opportunities in KRAS Wildtype Pancreatic Ductal Adenocarcinoma.

PURPOSE: To investigate the molecular characteristics of and potential for precision medicine in KRAS wildtype pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: We investigated 27 patients with KRASWT PDAC at our institution. Clinical data were obtained via chart review. Tumor specimens for each subject were interrogated for somatic single nucleotide variants, insertion and deletions, and copy number variants by DNA sequencing. Gene fusions were detected from RNA-seq. A patient-derived organoid (PDO) was developed from a patient with a MET translocation and expanded ex vivo to predict therapeutic sensitivity prior to enrollment in a phase 2 clinical trial. RESULTS: Transcriptomic analysis showed our cohort may be stratified by the relative gene expression of the KRAS signaling cascade. The PDO derived from our patient harboring a TFG-MET rearrangement was found to have in vitro sensitivity to the multi-tyrosine kinase inhibitor crizotinib. The patient was enrolled in the phase 2 SPARTA clinical trial and received monotherapy with vebrelitinib, a c-MET inhibitor, and achieved a partial and durable response. CONCLUSIONS: KRASWT PDAC is molecularly distinct from KRASMUT and enriched with potentially actionable genetic variants. In our study, transcriptomic profiling revealed that the KRAS signaling cascade may play a key role in KRASWT PDAC. Our report of a KRASWT PDAC patient with TFG-MET rearrangement who responded to a cMET inhibitor further supports the pursuit of precision oncology in this sub-population. Identification of targetable mutations, perhaps through approaches like RNA-seq, can help enable precision-driven approaches to select optimal treatment based on tumor characteristics.

Culture media composition influences patient-derived organoid ability to predict therapeutic responses in gastrointestinal cancers.

BACKGROUNDA patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO's ability to predict clinical response to gastrointestinal (GI) cancers.METHODSWe generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments.RESULTSWe report an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed a significant difference in response to standard-of-care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDO cultures.CONCLUSIONWhile we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers.TRIAL REGISTRATIONNot applicable.FUNDINGThe Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, University of Minnesota-Mayo Clinic Partnership, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).

Ecoevolutionary biology of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), the most common histological subtype of pancreatic cancer, is an aggressive disease predicted to be the 2nd cause of cancer mortality in the US by 2040. While first-line therapy has improved, 5-year overall survival has only increased from 5 to ∼10%, and surgical resection is only available for ∼20% of patients as most present with advanced disease, which is invariably lethal. PDAC has well-established highly recurrent mutations in four driver genes including KRAS, TP53, CDKN2A, and SMAD4. Unfortunately, these genetic drivers are not currently therapeutically actionable. Despite extensive sequencing efforts, few additional significantly recurrent and druggable drivers have been identified. In the absence of targetable mutations, chemotherapy remains the mainstay of treatment for most patients. Further, the role of the above driver mutations on PDAC initiation and early development is well-established. However, these mutations alone cannot account for PDAC heterogeneity nor discern early from advanced disease. Taken together, management of PDAC is an example highlighting the shortcomings of the current precision medicine paradigm. PDAC, like other malignancies, represents an ecoevolutionary process. Better understanding the disease through this lens can facilitate the development of novel therapeutic strategies to better control and cure PDAC. This review aims to integrate the current understanding of PDAC pathobiology into an ecoevolutionary framework.

Oncogenic gene expression and epigenetic remodeling of cis-regulatory elements in ASXL1-mutant chronic myelomonocytic leukemia.

Myeloid neoplasms are clonal hematopoietic stem cell disorders driven by the sequential acquisition of recurrent genetic lesions. Truncating mutations in the chromatin remodeler ASXL1 (ASXL1MT) are associated with a high-risk disease phenotype with increased proliferation, epigenetic therapeutic resistance, and poor survival outcomes. We performed a multi-omics interrogation to define gene expression and chromatin remodeling associated with ASXL1MT in chronic myelomonocytic leukemia (CMML). ASXL1MT are associated with a loss of repressive histone methylation and increase in permissive histone methylation and acetylation in promoter regions. ASXL1MT are further associated with de novo accessibility of distal enhancers binding ETS transcription factors, targeting important leukemogenic driver genes. Chromatin remodeling of promoters and enhancers is strongly associated with gene expression and heterogenous among overexpressed genes. These results provide a comprehensive map of the transcriptome and chromatin landscape of ASXL1MT CMML, forming an important framework for the development of novel therapeutic strategies targeting oncogenic cis interactions.

Research on Anal Squamous Cell Carcinoma: Systemic Therapy Strategies for Anal Cancer.

Anal squamous cell carcinoma (ASCC) is a rare malignancy, with most cases associated with human papilloma virus and an increased incidence in immunocompromised patients. Progress in management of ASCC has been limited not only due to its rarity, but also the associated lack of research funding and social stigma. Historically, standard of care for invasive ASCC has been highly morbid surgical resection, requiring a permanent colostomy. Surgery was associated with disease recurrence in approximately half of the patients. However, the use of chemotherapy (5-fluorouracil and mitomycin C) concomitantly with radiation in the 1970s resulted in disease regression, curing a subset of patients and sparing them from morbid surgery. Validation of the use of systemic therapy in prospective trials was not achieved until approximately 20 years later. In this review, advancements and shortcomings in the use of systemic therapy in the management of ASCC will be discussed. Not only will standard-of-care systemic therapies for locoregional and metastatic disease be reviewed, but the evolving role of novel treatment strategies such as immune checkpoint inhibitors, HPV-based vaccines, and molecularly targeted therapies will also be covered. While advances in ASCC treatment have remained largely incremental, with increased biological insight, an increasing number of promising systemic treatment modalities are being explored.

RAS mutations drive proliferative chronic myelomonocytic leukemia via a KMT2A-PLK1 axis.

Proliferative chronic myelomonocytic leukemia (pCMML), an aggressive CMML subtype, is associated with dismal outcomes. RAS pathway mutations, mainly NRASG12D, define the pCMML phenotype as demonstrated by our exome sequencing, progenitor colony assays and a Vav-Cre-NrasG12D mouse model. Further, these mutations promote CMML transformation to acute myeloid leukemia. Using a multiomics platform and biochemical and molecular studies we show that in pCMML RAS pathway mutations are associated with a unique gene expression profile enriched in mitotic kinases such as polo-like kinase 1 (PLK1). PLK1 transcript levels are shown to be regulated by an unmutated lysine methyl-transferase (KMT2A) resulting in increased promoter monomethylation of lysine 4 of histone 3. Pharmacologic inhibition of PLK1 in RAS mutant patient-derived xenografts, demonstrates the utility of personalized biomarker-driven therapeutics in pCMML.

Salicylates enhance CRM1 inhibitor antitumor activity by induction of S-phase arrest and impairment of DNA-damage repair.

Chromosome region maintenance protein 1 (CRM1) mediates protein export from the nucleus and is a new target for anticancer therapeutics. Broader application of KPT-330 (selinexor), a first-in-class CRM1 inhibitor recently approved for relapsed multiple myeloma and diffuse large B-cell lymphoma, have been limited by substantial toxicity. We discovered that salicylates markedly enhance the antitumor activity of CRM1 inhibitors by extending the mechanisms of action beyond CRM1 inhibition. Using salicylates in combination enables targeting of a range of blood cancers with a much lower dose of selinexor, thereby potentially mitigating prohibitive clinical adverse effects. Choline salicylate (CS) with low-dose KPT-330 (K+CS) had potent, broad activity across high-risk hematological malignancies and solid-organ cancers ex vivo and in vivo. The K+CS combination was not toxic to nonmalignant cells as compared with malignant cells and was safe without inducing toxicity to normal organs in mice. Mechanistically, compared with KPT-330 alone, K+CS suppresses the expression of CRM1, Rad51, and thymidylate synthase proteins, leading to more efficient inhibition of CRM1-mediated nuclear export, impairment of DNA-damage repair, reduced pyrimidine synthesis, cell-cycle arrest in S-phase, and cell apoptosis. Moreover, the addition of poly (ADP-ribose) polymerase inhibitors further potentiates the K+CS antitumor effect. K+CS represents a new class of therapy for multiple types of blood cancers and will stimulate future investigations to exploit DNA-damage repair and nucleocytoplasmic transport for cancer therapy in general.

ST3GAL1 is a target of the SOX2-GLI1 transcriptional complex and promotes melanoma metastasis through AXL.

Understanding the molecular events controlling melanoma progression is of paramount importance for the development of alternative treatment options for this devastating disease. Here we report a mechanism regulated by the oncogenic SOX2-GLI1 transcriptional complex driving melanoma invasion through the induction of the sialyltransferase ST3GAL1. Using in vitro and in vivo studies, we demonstrate that ST3GAL1 drives melanoma metastasis. Silencing of this enzyme suppresses melanoma invasion and significantly reduces the ability of aggressive melanoma cells to enter the blood stream, colonize distal organs, seed and survive in the metastatic environment. Analysis of glycosylated proteins reveals that the receptor tyrosine kinase AXL is a major effector of ST3GAL1 pro-invasive function. ST3GAL1 induces AXL dimerization and activation that, in turn, promotes melanoma invasion. Our data support a key role of the ST3GAL1-AXL axis as driver of melanoma metastasis, and highlight the therapeutic potential of targeting this axis to treat metastatic melanoma.

E3 ubiquitin ligase PARK2, an inhibitor of melanoma cell growth, is repressed by the oncogenic ERK1/2-ELK1 transcriptional axis.

Malignant melanoma, the most aggressive form of skin cancer, is characterized by high prevalence of BRAF/NRAS mutations and hyperactivation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), mitogen-activated protein kinases (MAPK), leading to uncontrolled melanoma growth. Efficacy of current targeted therapies against mutant BRAF or MEK1/2 have been hindered by existence of innate or development of acquired resistance. Therefore, a better understanding of the mechanisms controlled by MAPK pathway driving melanogenesis will help develop new treatment approaches targeting this oncogenic cascade. Here, we identify E3 ubiquitin ligase PARK2 as a direct target of ELK1, a known transcriptional effector of MAPK signaling in melanoma cells. We show that pharmacological inhibition of BRAF-V600E or ERK1/2 in melanoma cells increases PARK2 expression. PARK2 overexpression reduces melanoma cell growth in vitro and in vivo and induces apoptosis. Conversely, its genetic silencing increases melanoma cell proliferation and reduces cell death. Further, we demonstrate that ELK1 is required by the BRAF-ERK1/2 pathway to repress PARK2 expression and promoter activity in melanoma cells. Clinically, PARK2 is highly expressed in WT BRAF and NRAS melanomas, but it is expressed at low levels in melanomas carrying BRAF/NRAS mutations. Overall, our data provide new insights into the tumor suppressive role of PARK2 in malignant melanoma and uncover a novel mechanism for the negative regulation of PARK2 via the ERK1/2-ELK1 axis. These findings suggest that reactivation of PARK2 may be a promising therapeutic approach to counteract melanoma growth.

GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

Targeting of the Hedgehog/GLI and mTOR pathways in advanced pancreatic cancer, a phase 1 trial of Vismodegib and Sirolimus combination.

BACKGROUND/OBJECTIVES: Preclinical data indicated a functional and molecular interaction between Hedgehog (HH)/GLI and PI3K-AKT-mTOR pathways promoting pancreatic ductal adenocarcinoma (PDAC). A phase I study was conducted of Vismodegib and Sirolimus combination to evaluate maximum tolerated dose (MTD) and preliminary anti-tumor efficacy. METHODS: Cohort I included advanced solid tumors patients following a traditional 3 + 3 design. Vismodegib was orally administered at 150 mg daily with Sirolimus starting at 3 mg daily, increasing to 6 mg daily at dose level 2. Cohort II included only metastatic PDAC patients. Anti-tumor efficacy was evaluated every two cycles and target assessment at pre-treatment and after a single cycle. RESULTS: Nine patient were enrolled in cohort I and 22 patients in cohort II. Twenty-eight patients were evaluated for dose-limiting toxicities (DLTs). One DLT was observed in each cohort, consisting of grade 2 mucositis and grade 3 thrombocytopenia. The MTD for Vismodegib and Sirolimus were 150 mg daily and 6 mg daily, respectively. The most common grade 3-4 toxicities were fatigue, thrombocytopenia, dehydration, and infections. A total of 6 patients had stable disease. No partial or complete responses were observed. Paired biopsy analysis before and after the first cycle in cohort II consistently demonstrated reduced GLI1 expression. Conversely, GLI and mTOR downstream targets were not significantly affected. CONCLUSIONS: The combination of Vismodegib and Sirolimus was well tolerated. Clinical benefit was limited to stable disease in a subgroup of patients. Targeting efficacy demonstrated consistent partial decreases in HH/GLI signaling with limited impact on mTOR signaling. These findings conflict with pre-clinical models and warrant further investigations.

It Takes a Village to Overcome KRAS Dependence in Pancreatic Cancer.

In this issue, Hou and colleagues present their exciting work demonstrating that, through remodeling of the local tumor microenvironment (TME), pancreatic ductal adenocarcinoma forms a tumor-supportive niche capable of liberating cancer cells from dependence on oncogenic KRAS signaling. Through extensive experimentation both in vitro and in vivo, the authors reveal that the HDAC5-CCL2 axis drives the recruitment of tumor-associated macrophages to the TME to provide trophic signaling.See related article by Hou et al., p. 1058.

The extracellular sulfatase SULF2 promotes liver tumorigenesis by stimulating assembly of a promoter-looping GLI1-STAT3 transcriptional complex.

The expression of the extracellular sulfatase SULF2 has been associated with increased hepatocellular carcinoma (HCC) growth and poor patient survival. However, the molecular mechanisms underlying SULF2-associated tumor growth remain unclear. To address this gap, here we developed a transgenic mouse overexpressing Sulf2 in hepatocytes under the control of the transthyretin promoter. In this model, Sulf2 overexpression potentiated diethylnitrosamine-induced HCC. Further analysis indicated that the transcription factor GLI family zinc finger 1 (GLI1) mediates Sulf2 expression during HCC development. A cross of the Sulf2-overexpressing with Gli1-knockout mice revealed that Gli1 inactivation impairs SULF2-induced HCC. Transcriptomic analysis revealed that Sulf2 overexpression is associated with signal transducer and activator of transcription 3 (STAT3)-specific gene signatures. Interestingly, the Gli1 knockout abrogated SULF2-mediated induction of several STAT3 target genes, including suppressor of cytokine signaling 2/3 (Socs2/3); Pim-1 proto-oncogene, Ser/Thr kinase (Pim1); and Fms-related tyrosine kinase 4 (Flt4). Human orthologs were similarly regulated by SULF2, dependent on intact GLI1 and STAT3 functions in HCC cells. SULF2 overexpression promoted a GLI1-STAT3 interaction and increased GLI1 and STAT3 enrichment at the promoters of their target genes. Interestingly, the SULF2 overexpression resulted in GLI1 enrichment at select STAT3 consensus sites, and vice versa. siRNA-mediated STAT3 or GLI1 knockdown reduced promoter binding of GLI1 and STAT3, respectively. Finally, chromatin-capture PCR confirmed long-range co-regulation of SOCS2 and FLT3 through changes in promoter conformation. These findings define a mechanism whereby SULF2 drives HCC by stimulating formation of a GLI1-STAT3 transcriptional complex.