Driver Signaling Pathways Research Articles

Abstract P6-11-02: Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis caused by a limited understanding of its driver signaling pathways. We used the transmitochondrial cybrid approach to understand the metabolic reprogramming and mitochondria-nuclear crosstalk in metastatic TNBC. In cybrids, mitochondria from cancer and benign cells are compared under a commonly defined nuclear background. Using cybrid-based discovery and its validation in cell lines, patient-derived xenografts, and clinical data, we have previously reported the activation of fatty acid oxidation (FAO) in metastatic TNBC. Recently, we developed a unique gene (DEG) signature by integrating gene expression data from cybrids with different omnibus databases such as TCGA and METABRIC. Our DEG could effectively distinguish the PAM50 subtypes of BC. Further analyses nominated negative regulation of the canonical WNT signaling pathway as one of the major pathways altered by mitochondrial character. The WNT pathway is known to regulate cell signaling, metabolism, epithelial-mesenchymal transition (EMT), and cancer stemness. Cybrids with benign mitochondria showed transcriptional activation of negative WNT regulators, including DKK1, SOST, DAB2, and CAV1. Citrate is a key metabolite from the tricarboxylic acid cycle (TCA cycle) and is an intermediate of many other metabolic pathways. Analysis of TNBC cybrids, cell lines, and BC tissues suggest that in TNBC, citrate levels are increased with the activation of CPT1A, the rate-limiting enzyme of FAO. We confirmed the reduction of the beta-catenin protein in TNBC cells after pharmacological and genetic inhibition of CPT1A and mitochondrial citrate transporter (SLC25A1). We further analyzed the gene set enrichment analysis (GSEA) using gene expression data developed from short and long-term CPT1A inhibited TNBC cells and observed that the FAO alters polycomb repressive complex 2 (PRC2) activity. PRC2 is a critical modulator of the H3K27 methylation pattern as well as pathways related to cancer stemness, including WNT/beta-catenin and Hedgehog (SHH) pathways. These results suggest that the mitochondrial FAO transcriptionally inhibits WNT antagonist genes through repressive histone modification and consequently activates the canonical WNT pathway and cancer stemness. Overall, our current observations provide new insights into the regulation WNT pathway by mitochondria-nuclear crosstalk in TNBC. Citation Format: Sukjin Yang, Jun Hyoung Park, Suna Kim, Meron Ghidey, Benny Kaipparettu. Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-02.

Identification of genes and pathways leading to poor prognosis of non-small cell lung cancer using integrated bioinformatics analysis.

BackgroundNon-small cell lung cancer (NSCLC) is a common malignancy with a high morbidity and mortality rate worldwide, but the driver genes and signaling pathways involved are largely unclear. Herein, our study aimed to identify significant genes with poor outcome and underlying mechanisms in NSCLC using bioinformatics analyses.MethodsGene expression profiles (GSE33532, GSE19188, GSE102287, GSE27262), including 319 NSCLC and 232 adjacent lung tissues, were downloaded from the GEO database. Differentially expressed genes (DEGs) were identified by the GEO2R online tool. Functional and pathway enrichment analyses were performed via the DAVID database. The protein-protein interactions (PPIs) of these DEGs were constructed by the STRING website and visualized by the Cytoscape software platform. The expression of hub genes in NSCLC was validated through the GEPIA database. Kaplan-Meier plotter was used to analyse the survival rate with multivariate Cox regression. The expression of protein tyrosine kinase 2 (PTK2) in NSCLC and adjacent lung tissues was evaluated on the UALCAN database platform.ResultsA total of 225 significant DEGs were obtained between NSCLC and adjacent lung tissues, containing 52 upregulated genes and 173 downregulated genes. The DEGs were clustered based on functions and signaling pathways that may be closely associated with NSCLC occurrence. A total of 174 DEGs were identified from the PPI network complex. Top 10 hub genes were selected by CytoHubba plugin. As independent predictors, seven genes (COL1A1, ADAM12, VWF, OGN, EDN1, CAV1, ITGA8) were associated with poor prognosis in NSCLC via multivariate Cox regression (P<0.01). Four genes (VWF, CAV1, ITGA8, COL1A1) were found to be significantly enriched in the focal adhesion pathway (P=1.04E-04) and to be upstream regulators of PTK2. PTK2 was upregulated in NSCLC and associated with poor survival prognosis in lung squamous cell carcinoma (LUSC).ConclusionsTaken together, the important genes and pathways in NSCLC were identified by using integrated bioinformatics analysis. PTK2 could be a key gene associated with the biological process of NSCLC formation and progression and a potential therapeutic target for NSCLC treatment.

Relationship of PI3K-Akt/mTOR/AMPK signaling pathway genetic mutation with efficacy and prognosis in nasopharyngeal carcinoma.

Genetic mutation is one of the important causes for tumor genesis and development, but genetic mutation in nasopharyngeal carcinoma (NPC) has rarely been reported. This study explored the role of phosphatidylinositol 3 kinase-protein kinase B (PI3K-Akt), mammalian target of rapamycin (mTOR), and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathway in the efficacy and prognosis in patients with NPC. A total of 31 patients with advanced NPC, who came from the Affiliated Cancer Hospital of Xiangya School of Medicine of Central South University/Hunan Provincial Cancer Hospital, were enrolled. All of the exons of 288 genes, introns of 38 genes and promoters or fusion breakpoint regions from the nasopharyngeal biopsy tissues before treatment were detected by the gene sequencing platform Illumina NextSeq CN500. The coding regions of 728 genes were carried out a high-depth sequencing of target region capture, and the 4 variant types of tumor genes (including point mutations, insertion deletions of small fragments, copy number variations, and currently known fusion genes) were detected. All of 31 patients received platinum-based induction chemotherapy combined with concurrent chemoradiotherapy and were followed up for a long time. The 3-year regional failure-free survival (RFFS) and disease-free survival (DFS) in patients with PI3K-Akt pathway mutation were significantly lower than those in unmutated patients (χ2=6.647, P<0.05). The 3-year RFFS and DFS in patients with mTOR pathway mutations were significantly lower than those in unmutated patients, and there was significant difference (χ2=5.570, P<0.05). The rate of complete response (CR) in patients with unmutated AMPK pathway was significantly higher than that in patients with mutation at 3 months after treatment (P<0.05), and the 3-year RFFS and DFS in patients with AMPK pathway mutation were significantly lower than those in unmutated patients (χ2=4.553, P<0.05). PI3K-Akt/mTOR/AMPK signaling pathway mutations and pre-treatment EB virus DNA copy numbers were independent prognostic factors for 3-year RFFS and DFS in patients with NPC (both P<0.05). The NPC patients with PI3K-Akt/mTOR/AMPK signaling pathway mutation have poor prognosis, and the detection of PI3K-Akt, mTOR, AMPK driver genes and signaling pathways by next-generation sequencing is expected to provide new idea for basic research and targeted therapy of NPC.

Abstract 2397: Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis, attributed to the limited understanding of its driver signaling pathways. Although TNBC exhibits unique characteristics, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are essential to reduce its severity. We have previously shown that metabolic reprogramming to fatty acid β-oxidation (FAO) is a dominant metabolic pathway in TNBC. We have also reported that FAO regulates c-Src, one of the frequently upregulated oncopathways in TNBC, via autophosphorylation of Src at Y419. However, single drug therapy using FAO inhibitors or Src inhibitors did not show a significant therapeutic advantage in TNBC. Biguanide such as metformin is one of the most widely administered anti-diabetic agents. Recently increasing evidence demonstrates that biguanide therapy can have an anticancer effect in various cancer types, including BC. Phenformin, a biguanide derivative similar to metformin, has greater potency than metformin. Both biguanides can suppress mitochondrial electron transport chain (ETC) function because of its inhibitory effect on ETC complex I activity. However, biguanides also lead to the activation of AMPK, which plays a vital role in insulin signaling and energy-sensing. Notably, AMPK is an upstream regulator of the FAO pathway because it can phosphorylate ACC and activate FAO. Considering the dependency of TNBC on mitochondrial FAO and the role of AMPK in the activation of FAO, our computational modeling predicted that the combined targeting of both ‘arms' of the pathway could provide a significant advantage to the anticancer effect in TNBC. Thus, we have used the combination of biguanides and FAO inhibitors to target both ETC and FAO respectively. Pharmacological and genetic approaches with multiple in vitro and in vivo analysis using TNBC cell lines and PDX models suggest that the combination of biguanides and FAO inhibitors can provide significant advantage in TNBC therapy. Further studies also show that the combination of biguanides with Src inhibitors can provide a better therapeutic option to target metastatic TNBC. We are currently evaluating these findings' translational significance as a cost-effective approach to managing non-targetable TNBCs. Citation Format: Junhyoung Park, Kwang Hwa Jung, Dongya Jia, Sukjin Yang, Dharaniya Sakthivel, Abha Tiwari, José N. Onuchic, Benny Abraham Kaipparettu. Significance of the combination of biguanides and fatty acid β-oxidation inhibitors in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2397.

Quantification of Plasma Proteins from Idiopathic Multicentric Castleman Disease Flares and Remissions Reveals ‘Chemokine Storm’ and Separates Clinical Subtypes

Introduction: Human Herpesvirus-8(HHV-8)-negative/idiopathic multicentric Castleman disease (iMCD) is a poorly understood disease involving polyclonal lymphoproliferation, constitutional symptoms, and life-threatening multi-organ failure. Some patients experience thrombocytopenia, anasarca, myelofibrosis, renal dysfunction, and organomegaly (TAFRO Syndrome) while others (non-TAFRO) experience thrombocytosis and hypergammaglobulinemia. Though the etiology is unknown, iMCD symptoms and disease progression are believed to be driven by a cytokine storm, often including interleukin-6 (IL-6). Anti-IL-6 therapy with siltuximab was approved by the FDA to treat iMCD based on superior efficacy compared to placebo in a randomized trial. However, approximately one-half of iMCD patients do not respond to anti-IL-6 therapy. Few rational treatment options exist for anti-IL-6 non-responders, because alternative driver cytokines and signaling pathways are not known. Furthermore, no positive diagnostic biomarkers exist for iMCD. We systematically characterized proteomic changes that occur in iMCD patients between flare and remission to identify key cytokines, signaling pathways, and cell types that may be involved in pathogenesis.Methods: Using Somalogic's modified aptamer-based proteomics platform, SOMAScan, we quantified 1,129 plasma proteins from six iMCD patients at the University of Arkansas for Medical Sciences during flare and remission. Two cases were confirmed to have TAFRO syndrome, and four cases did not have TAFRO syndrome. Proteomic data for two sets of healthy controls were obtained post-hoc and merged with the iMCD proteomic data along common calibrated proteins. Data analysis was performed using R 3.3.3.Results: Principal component analysis (PCA) revealed a distinction between iMCD samples-in flare and remission-and healthy control samples. Reactome pathway analyses identified cytokine signaling, chemokine signaling, and the complement cascade as the most enriched pathways compared to the background proteins assayed. Chemokines represented the greatest proportion of two-fold up-regulated cytokines in flare compared to interleukins and other cytokines. The chemokine, B Lymphocyte Chemoattractant (BLC)/CXCL13, was the most significantly upregulated cytokine across all patients. IL-6 was surprisingly not among the most upregulated cytokines. Five of six patients had less than two-fold up-regulation and the only patient with greater than two-fold up-regulation did not improve with anti-IL-6 receptor monotherapy. However, three of the other four patients treated with anti-IL-6 or anti-IL-6 receptor monotherapy experienced responses despite small changes in IL-6 levels between flare and remission. We observed a distinct proteomic profile between the TAFRO and non-TAFRO patients according to PCA, correlation analyses, and hierarchical clustering. The two TAFRO patients also both failed to respond to anti-IL-6 monotherapy. A large proportion of proteins significantly up- or down-regulated in non-TAFRO patients were conversely up- or down-regulated in TAFRO patients.Discussion: This study provides the first unbiased quantification of circulating proteins in iMCD patients during flare and remission. PCA of these data suggest that the proteomic profile of iMCD patients differs from that of healthy individuals even during remission. Our results support the field's assertion that iMCD involves a cytokine storm, but increased IL-6 was not uniformly present. The response of three patients to anti-IL-6 treatment suggests that IL-6 plays a critical role in these patients despite subtle changes in IL-6 levels. The increased proportion of up-regulated chemokines relative to other cytokines suggests that iMCD involves a chemokine storm. CXCL13 plays an essential role in honing B cells to germinal centers, which are uniformly dysmorphic in iMCD with either too few (atrophic) or too many B cells (hyperplastic). Lymph node stromal cells are known to produce three of the most up-regulated chemokines (CXCL13, CCL19 and CCL21), suggesting that they may play a role in pathogenesis. The distinct proteomic profiles between TAFRO (both anti-IL-6 non-responders) and non-TAFRO (responders) cases suggest that differing mechanisms may be involved. Larger studies are needed to confirm these findings and investigate chemokines in iMCD pathogenesis. DisclosuresFajgenbaum:Janssen Pharmaceuticals: Research Funding.

Combined Inhibition of Gαq and MEK Enhances Therapeutic Efficacy in Uveal Melanoma.

All uveal melanoma and a fraction of other melanoma subtypes are driven by activation of the G-protein alpha-q (Gαq) pathway. Targeting these melanomas has proven difficult despite advances in the molecular understanding of key driver signaling pathways in the disease pathogenesis. Inhibitors of Gαq have shown promising preclinical results, but their therapeutic activity in distinct Gαq mutational contexts and in vivo have remained elusive. We used an isogenic melanocytic cellular system to systematically examine hotspot mutations in GNAQ (e.g., G48V, R183Q, Q209L) and CYSLTR2 (L129Q) found in human uveal melanoma. This cellular system and human uveal melanoma cell lines were used in vitro and in in vivo xenograft studies to assess the efficacy of Gαq inhibition as a single agent and in combination with MEK inhibition. We demonstrate that the Gαq inhibitor YM-254890 inhibited downstream signaling and in vitro growth in all mutants. In vivo, YM-254890 slowed tumor growth but did not cause regression in human uveal melanoma xenografts. Through comprehensive transcriptome analysis, we observed that YM-254890 caused inhibition of the MAPK signaling with evidence of rebound by 24 hours and combination treatment of YM-254890 and a MEK inhibitor led to sustained MAPK inhibition. We further demonstrated that the combination caused synergistic growth inhibition in vitro and tumor shrinkage in vivo. These data suggest that the combination of Gαq and MEK inhibition provides a promising therapeutic strategy and improved therapeutic window of broadly targeting Gαq in uveal melanoma.See related commentary by Neelature Sriramareddy and Smalley, p. 1217.

Identification of Driver Genes Regulating the T-Cell-Infiltrating Levels in Hepatocellular Carcinoma.

The driver genes regulating T-cell infiltration are important for understanding immune-escape mechanisms and developing more effective immunotherapy. However, researches in this field have rarely been reported in hepatocellular carcinoma (HCC). In the present study, we identified cancer driver genes triggered by copy number alterations such as CDKN2B, MYC, TSC1, TP53, and GSK3B. The T-cell infiltration levels were significantly decreased in both HCC and recurrent HCC tissues compared with the adjacent normal liver tissues. Remarkably, we identified that copy number losses of MAX and TP53 were candidate driver genes that significantly suppress T-cell infiltration in HCC. Accordingly, their downstream oncogenic pathway, cell cycle, was significantly activated in the low T-cell infiltration HCC. Moreover, the chemokine-related target genes by TP53, which played key roles in T-cell recruitment, were also downregulated in HCC with TP53/MAX deletions, suggesting that copy number losses in MAX and TP53 might result in T-cell depletion in HCC via downregulating chemokines. Clinically, the T-cell infiltration levels and chemokines activity could accurately predict the response of sorafenib, and the prognostic outcomes in HCC. In conclusion, the systematic analysis not only facilitates identification of driver genes and signaling pathways involved in T-cell infiltration and immune escape, but also gains more insights into the functional roles of T cells in HCC.

Abstract 1331: First results of the EIT PACMAN Study: OncoSignal pathway analysis to identify clinically actionable signal transduction pathway activity in a variety of cancer types

Abstract Introduction: Precision medicine has shown to improve outcomes of cancer patients by identifying oncogenic alterations and actionable mutations. Yet, most tests perform DNA sequencing and are not able to discriminate which tumor driving signaling pathways (SP) are functionally active. OncoSignal pathway analysis tests quantitatively measure activity of SP such as estrogen receptor, androgen receptor, PI3K, MAPK, TGF-β, Notch on fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples, while mutation analysis provides complementary information related to the genomic alteration in the SP. Combined information is expected to improve choice of the optimal effective targeted therapy to improve patients' outcome and quality of life as well as to reduce unnecessary side effects/costs due to unnecessary therapy. In this study OncoSignal pathway analysis was performed on a series of samples from the Moscato trial (1), with the aim of assessing clinically actionable SP activity. Methods: OncoSignal pathway analysis (ER, AR, PI3K, MAPK, HH, Notch, TGF-β) was performed blinded by Molecular Pathway Dx (Philips, Eindhoven) on tumor tissue samples from 5 breast and 31 prostate tumors, all proven to be hard to treat and obtained from the Moscato study. Results were sent back to Institut Gustave Roussy for clinical annotation and analysis. For breast and prostate cancer, pathway activity scores were determined for each SP in healthy breast (n=7) and prostate (n=9) tissues (GEO datasets: GSE17951 and GSE10780) and compared with pathway activity scores in cancer tissue samples (multiple GEO datasets: n=133 and n=1712). Increased activity of a pathway in cancer tissue (&amp;gt;95th percentile of healthy tissue pathway activity) was considered as tumor-driver function for the respective pathway and thus clinically actionable. Subsequently, for each individual Moscato sample, alterations were considered as tumor driving pathways if the sample pathway activity score exceeded the 95th percentile of normal (primary) tissue pathway activity. Results: Identified tumor driving SP in breast cancer were ER, AR, MAPK-AP1, HH, and PI3K pathway whereas in prostate cancer identified SP were the AR and PI3K pathway. Of the 5 breast cancer metastasis samples (lung, head/neck, skin, liver, lymph node; average tumor content 40%), all had at least one clinically actionable tumor driving pathway. Of the 30 prostate cancer metastasis samples (bone, liver, lymph nodes; average tumor content 62%), 29 (97%) had a clinically actionable tumor driving pathway. Conclusion: OncoSignal pathway analysis enabled identification of clinically potentially actionable signaling pathway activity in 97-100% of analyzed breast and prostate cancer samples. Citation Format: Patricia Martin-Romano, Sieglinde Neerken, Anja Van de Stolpe, Eveline Den Biezen-Timmerman, Maud Ngo, Claudio Nicotra, Martijn Akse, Alexander Eggermont, Paul Van de Wiel, Christophe Massard, Fabien Calvo. First results of the EIT PACMAN Study: OncoSignal pathway analysis to identify clinically actionable signal transduction pathway activity in a variety of cancer types [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 1331.

EIT PACMAN Study preliminary results: OncoSignal pathway analysis identifies actionable cancer targets.

e15606 Background: Precision medicine refers to tailoring of treatment to each individual patient, although identifying tumor driving signaling pathways (SP) that are functionally active is still a challenge. OncoSignal pathway tests quantitatively measure activity of SP such as estrogen receptor (ER), androgen receptor (AR), PI3K, MAPK, Hedgehog (HH), TGF-β, Notch on fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue samples. OncoSignal pathway analysis aimed at assessing clinically actionable SP and retrospectively predicting targeted drug response on a series of patients’ (pts) samples from the MOSCATO trial run at Gustave Roussy. Methods: OncoSignal pathway analysis (ER, AR, PI3K, MAPK, HH, Notch, TGF-β) was performed blinded by Molecular Pathway Dx (Philips, Eindhoven) on metastatic tumor tissue samples from breast cancer (BC), prostate (PC), and high grade serous ovarian cancers (OC). Using Affymetrix expression array data from public GEO datasets, SP activity was analyzed in healthy prostate, breast, and ovarian tissue to define abnormal SP activity thresholds for tumor tissue pathway analysis. For each individual sample, SP alterations were considered tumor driving SP if sample SP activity exceeded the 95th percentile of SP activity within healthy tissue. Results by OncoSignal were also combined with clinical characteristics and molecular alterations identified in the MOSCATO trial. Results: Identified tumor driving SP were ER, AR, MAPK-AP1, HH, PI3K pathway in BC (n = 5), AR in PC (n = 30); AP1, Notch, TGFβ in OC (n = 17). OncoSignal identified clinically actionable tumor driving pathways in all BC samples (median tumor cellularity [MTC]: 40%, range 15-80%); 30/31 PC samples (MTC: 62%, range 25-90%), 16/17 OC samples (MTC 62%, range 15-80%). Actionable mutations were previously identified in 4/5 BC; 13/31 PC; 6/17 OC. Seven pts with BC and PC were treated with targeted therapy. OncoSignal pathway analysis correctly predicted response/resistance in 4 of these pts (57%). Conclusions: OncoSignal pathway analysis correctly identified SP activity alterations and predicted targeted drug response in this series of patients. OncoSignal will be further validated prospectively in precision medicine studies at Gustave Roussy in which patients are stratified for targeted treatment by mutation analysis.

SMYD3: An Oncogenic Driver Targeting Epigenetic Regulation and Signaling Pathways.

SMYD3 is a member of the SMYD lysine methylase family and plays an important role in the methylation of various histone and non-histone targets. Aberrant SMYD3 expression contributes to carcinogenesis and SMYD3 upregulation was proposed as a prognostic marker in various solid cancers. Here we summarize SMYD3-mediated regulatory mechanisms, which are implicated in the pathophysiology of cancer, as drivers of distinct oncogenic pathways. We describe SMYD3-dependent mechanisms affecting cancer progression, highlighting SMYD3 interplay with proteins and RNAs involved in the regulation of cancer cell proliferation, migration and invasion. We also address the effectiveness and mechanisms of action for the currently available SMYD3 inhibitors. The findings analyzed herein demonstrate that a complex network of SMYD3-mediated cytoplasmic and nuclear interactions promote oncogenesis across different cancer types. These evidences depict SMYD3 as a modulator of the transcriptional response and of key signaling pathways, orchestrating multiple oncogenic inputs and ultimately, promoting transcriptional reprogramming and tumor transformation. Further insights into the oncogenic role of SMYD3 and its targeting of different synergistic oncogenic signals may be beneficial for effective cancer treatment.

A Phase 1 a/b Dose Escalation Trial to Evaluate the Safety and Tolerability of CG-806 in Patients with Relapsed or Refractory CLL/SLL or Non-Hodgkin's Lymphomas

INTRODUCTION: CG-806 is a first-in-class, oral, non-covalent, and potent inhibitor of the wild type and all known mutant forms of Bruton's tyrosine kinase (BTK, including the C481S mutation), as well as all forms of the FLT3 receptor tyrosine kinase and multiple oncogenic signaling pathways, but with a selectivity that spares normal cells by avoiding off-target inhibition of kinases known to produce toxicities. Such a kinase inhibitory profile is key to the treatment of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL) and certain B-cell malignancies, which are driven by dysregulated BTK, can become resistant to covalent BTK inhibitors (ibrutinib and others) by BTK C481S mutation, and may become refractory to various therapies by other compensatory kinase signaling pathways such as SYK, SRC, STATs, ERK, AKT, NFĸB and MYC. In cell lines and primary samples from CLL and B-cell ALL patients, CG-806 potently suppressed the driver and compensatory pathways (inhibited phosphorylation of BTK, FLT3, PDGFRα, SYK, SRC, ERK, STAT5 and AKT, decreased MYC levels), induced cleavage of caspase 3, potently killed cells insensitive to ibrutinib or venetoclax, and caused enhanced cell killing in combination with venetoclax. Consequently, CG-806 is currently being evaluated in a Phase I a/b trial in patients with CLL/SLL or non-Hodgkin's lymphomas (NHLs). METHODS: A Phase 1 a/b trial of CG-806 in patients with CLL/SLL or non-Hodgkin's lymphomas (NCT03893682) is underway to evaluate the safety, tolerability and potential effectiveness of CG-806 for the treatment of patients for which either the standard treatment has failed, is no longer effective, or can no longer be administered safely. The starting dose is 150 mg PO BID and the dose will be escalated in 150 mg increments with only 1 patient at the two lowest dose levels and a minimum of 3 at the higher dose levels. Once the maximum tolerated dose or recommended dose is reached, up to 100 patients will be enrolled in the expansion cohort at the recommended dose. Primary objectives of the trial are to determine the safety and tolerability of CG-806 at the dose given orally every 12 hours that maintains a biologically active plasma concentration over a period of 28 days and to establish the recommended Phase 2 dose (RP2D) of CG-806 for future clinical trials in patients with advanced CLL/SLL or NHL. Key secondary objectives are to observe patients for any evidence antitumor activity using FDG PET-CT imaging evaluations and to measure pharmacodynamic biomarkers of drug effect including phospho-BTK levels and selected mRNA levels in peripheral white blood cells, as well as CCL3 and CCL4 circulating levels. RESULTS: CG-806 oral capsules have been administered at a starting dose of 150 mg BID on a 28-day cycle to the first CLL patient who previously failed ibrutinib, venetoclax, rituximab, idelalisib and other medications. To date (as of July 2019), in this first patient who has received more than 40 doses of CG-806 150mg oral capsules, no dose limiting toxicities have been observed, there are no impediments to continuing dosing on study, and the tolerability is consistent with preclinical tolerability findings in rodents and dogs when CG-806 was delivered as an oral gavage. Safety and tolerance vigilance, as well as pharmacokinetic (PK) and pharmacodynamic analyses, will continue into subsequent cycles and dose levels. CONCLUSIONS: CG-806 is an oral, non-covalent pan-BTK/pan-FLT3 inhibitor designed to suppress multiple oncogenic driver and compensatory signaling pathways in order to directly and potently kill B-cell cancer cells including those insensitive to the ibrutinib covalent BTK inhibitor and the venetoclax Bcl-2 inhibitor. Recruitment and enrollment of patients with R/R CLL/SLL and non-Hodgkin's lymphomas are continuing in a Phase 1a/b dose escalation/expansion trial, and updated safety, PK, and available biomarker data will be presented at the meeting. Disclosures Howell: Aptose Biosciences, Inc: Consultancy, Research Funding. Zhang:Aptose Biosciences, Inc: Employment. Rice:Aptose Biosciences, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Driver gene alterations and activated signaling pathways toward malignant progression of gastrointestinal stromal tumors.

Mutually exclusive KIT and PDGFRA mutations are considered to be the earliest events in gastrointestinal stromal tumors (GIST), but insufficient for their malignant progression. Herein, we aimed to identify driver genes and signaling pathways relevant to GIST progression. We investigated genetic profiles of 707 driver genes, including mutations, gene fusions, copy number gain or loss, and gene expression for 65 clinical specimens of surgically dissected GIST, consisting of six metastatic tumors and 59 primary tumors from stomach, small intestine, rectum, and esophagus. Genetic alterations included oncogenic mutations and amplification‐dependent expression enhancement for oncogenes (OG), and loss of heterozygosity (LOH) and expression reduction for tumor suppressor genes (TSG). We assigned activated OG and inactivated TSG to 27 signaling pathways, the activation of which was compared between malignant GIST (metastasis and high‐risk GIST) and less malignant GIST (low‐ and very low‐risk GIST). Integrative molecular profiling indicated that a greater incidence of genetic alterations of driver genes was detected in malignant GIST (96%, 22 of 23) than in less malignant GIST (73%, 24 of 33). Malignant GIST samples groups showed mutations, LOH, and aberrant expression dominantly in driver genes associated with signaling pathways of PI3K (PIK3CA, AKT1, and PTEN) and the cell cycle (RB1, CDK4, and CDKN1B). Additionally, we identified potential PI3K‐related genes, the expression of which was upregulated (SNAI1 and TPX2) or downregulated (BANK1) in malignant GIST. Based on our observations, we propose that inhibition of PI3K pathway signals might potentially be an effective therapeutic strategy against malignant progression of GIST.

Driver network as a biomarker: systematic integration and network modeling of multi-omics data to derive driver signaling pathways for drug combination prediction.

MotivationDrug combinations that simultaneously suppress multiple cancer driver signaling pathways increase therapeutic options and may reduce drug resistance. We have developed a computational systems biology tool, DrugComboExplorer, to identify driver signaling pathways and predict synergistic drug combinations by integrating the knowledge embedded in vast amounts of available pharmacogenomics and omics data.ResultsThis tool generates driver signaling networks by processing DNA sequencing, gene copy number, DNA methylation and RNA-seq data from individual cancer patients using an integrated pipeline of algorithms, including bootstrap aggregating-based Markov random field, weighted co-expression network analysis and supervised regulatory network learning. It uses a systems pharmacology approach to infer the combinatorial drug efficacies and synergy mechanisms through drug functional module-induced regulation of target expression analysis. Application of our tool on diffuse large B-cell lymphoma and prostate cancer demonstrated how synergistic drug combinations can be discovered to inhibit multiple driver signaling pathways. Compared with existing computational approaches, DrugComboExplorer had higher prediction accuracy based on in vitro experimental validation and probability concordance index. These results demonstrate that our network-based drug efficacy screening approach can reliably prioritize synergistic drug combinations for cancer and uncover potential mechanisms of drug synergy, warranting further studies in individual cancer patients to derive personalized treatment plans.Availability and implementationDrugComboExplorer is available at https://github.com/Roosevelt-PKU/drugcombinationprediction.Supplementary information Supplementary data are available at Bioinformatics online.

Serum Proteomics Reveals Distinct Subtypes Associated with Treatment Response in Idiopathic Multicentric Castleman Disease

Human herpesvirus-8(HHV-8)-negative/idiopathic multicentric Castleman disease (iMCD) is a rare and poorly understood disorder diagnosed in ~1,000 individuals in the USA each year. It involves polyclonal lymphoproliferation, constitutional symptoms, systemic inflammation and an uncontrollable cytokine storm resulting in life-threatening multi-organ failure. Diagnosis and treatment can be difficult due to limited etiological understanding and heterogeneous presentation - clinical, laboratory, and histopathological abnormalities overlap with infectious, autoimmune and oncological diseases. iMCD symptoms and disease progression are largely believed to be driven by interleukin-6 (IL-6). However, approximately 66% of iMCD patients did not respond to anti-IL-6 therapy, siltuximab, the only FDA-approved iMCD therapy, in its phase II study (NCT01024036). Few treatment options exist for anti-IL-6 refractory patients because alternative driver cytokines and signaling pathways are not known. Herein we report the largest study to-date of iMCD serum proteomes with correlative anti-IL6 response data from 92 iMCD patients in disease flare (n=75 of which were collected as part of NCT01024036), in order to: (1) molecularly define iMCD, (2) identify predictors of response to anti-IL6 therapy, and (3) gain insights into the pathogenesis of iMCD. Proteomes of HHV8-positive MCD (n=20), Hodgkin lymphoma (n=20), rheumatoid arthritis (n=20) and healthy individuals (n=44) were also analyzed. Of the ~1,300 analytes measured using SomaLogic SOMAscan, 1,178 passed QC and were included in analyses. Each analyte was log2 transformed and capped at the 2.5th and 97.5th percentiles. Clinical and laboratory data collected at the time of sample draw were used to calculate disease activity following a modified CHAP scale: C-reactive protein, hemoglobin and albumin; missing performance status. Response to siltuximab was determined in NCT01024036. Data analysis was performed using the Medidata Rave Omics machine learning platform and R v3.4.4. Clustering of baseline proteomic data for iMCD patients identified six clusters that ranged in size from seven to 27 subjects. No associations with race, site, sex, age, or batch were found. Analytes identified among the strongest differentiators include cytokines, chemokines and inflammatory molecules. Interestingly, the largest cluster was associated with response to siltuximab (p<0.05; 65% (11/17) vs 19% (5/27) in all others), higher disease activity (p<0.01), and higher IL-6 levels (p<0.01). Analysis of data for the entire study population separated HHV-8-positive MCD, Hodgkin lymphoma, and rheumatoid arthritis into distinct clusters. Of note, iMCD patients did not form a single or unique cluster, reinforcing the heterogeneity of the disease, and a subset of iMCD patients demonstrated similar, but not overlapping, proteomic profiles to those of Hodgkin lymphoma. These results indicate that previously undiscovered proteomically-distinct iMCD subtypes or disease states exist, which can be used to inform treatment options. Given that iMCD may have a sudden and severe onset, predictive markers of anti-IL6 therapy response are critical for timely administration of the correct treatment. Additionally, overlapping proteomic profiles of a subset of iMCD patients with Hodgkin lymphoma provide etiological insights. More broadly, this study represents the first use of high-quality serum proteomics data to study a rare non-malignant lymphoproliferative disorder and to assist with molecularly defining a heterogeneous disease, developing candidate diagnostic biomarkers, predicting response or failure to therapy, and identifying novel candidate therapeutic targets. DisclosuresGuilfoyle:Janssen Pharmaceuticals, Inc.: Employment. Tendler:Janssen Pharmaceuticals, Inc.: Employment, Equity Ownership. Reddy:Janssen Pharmaceuticals, Inc.: Employment, Equity Ownership. Weinblatt:Amgen: Consultancy, Research Funding; Sanofi/Regeneron: Consultancy, Research Funding; Crescendo Bioscience: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; UCB Pharmaceuticals: Consultancy, Research Funding. Bower:Merck: Honoraria; ViiV: Honoraria; Gilead: Honoraria; Janssen Pharmaceuticals: Honoraria. Masaki:Ono: Research Funding; Phizer: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Kyowa Hakko Kirin: Research Funding. Beineke:Medidata Solutions: Employment, Equity Ownership. Miljovska:Medidata Solutions: Employment. Katz:Medidata Solutions: Employment. Shenoy:Medidata Solutions: Consultancy. Oromendia:Medidata Solutions: Employment, Equity Ownership. Mezey:Medidata Solutions: Consultancy. Wiser:Medidata Solutions: Employment, Equity Ownership. Benbanaste:Medidata Solutions: Employment, Equity Ownership. Lee:Medidata Solutions: Employment. Fosså:Janssen Pharmaceuticals, Inc.: Honoraria. Fajgenbaum:Janssen Pharmaceuticals, Inc.: Research Funding.

Abstract 5029: Precision cancer medicine based on 3D drug profiling of patient-derived cancer cell spheroid models

Abstract We have set up a precision medicine strategy for solid tumors to i) understand biological heterogeneity and driver signaling pathways in cancer, ii) identify new drug opportunities, iii) develop biomarkers for drug responses, and iv) eventually tailor effective treatments for individual patients. Fresh cancer tissue is obtained directly from clinics and processed to provide patient -derived cells (PDCs). PDCs and original tumor tissues are characterized using genetic profiling and image-based phenotyping, phenomics. Systematic drug sensitivity and resistance testing (DSRT) is carried out on the representative PDC models. The PDCs are plated in 384-well plates and treated with oncological compounds, each in five concentrations in 384-well plates. The cell viability and toxicity are measured as drug responses using plate readers. Alternatively, drug-treated cells are immunostained and subjected to automated high-content imaging and image analysis. Central to the success of this approach is to grow and test the patient derived cells in ex vivo conditions mimicking the tumor microenvironment. To this aim, we have developed 3D drug profiling for PDC spheroids from ovarian and renal cancers. For the 3D drug profiling, cells were cultivated either in Matrigel or in cellulose-based hydrogel, GrowDex, which has earlier been shown to support 3D growth of cell lines and stem cells. The pipetting robot Biomek FXp (Beckman Coulter) was utilized for transfer of matrices and cells to 384-well plates, and acoustic dispenser Echo 550 (Labcyte) for delivery of tailored drug library of 52 drugs to the spheroids. The drug sensitivities of cancer cell spheroids were scored using cell viability measurement as well as high-content confocal imaging. Our results with primary cells and cell lines suggest that both Matrigel and cellulose-based hydrogel are applicable in the 384-well plate drug profiling assay, and PDC spheroids are formed in both conditions. When the drug responses of ovarian cancer PDCs grown in different 2D and 3D conditions were systematically compared, we observed significant differences in sensitivity to several drugs. Here, we describe the individual drug effects in all conditions, such as some chemotherapeutics being less effective in 3D. As a conclusion, the comparison of results from 2D and 3D drug profiling increases our understanding of mechanisms of drugs, and may aid to select the most representative drugs for the patient. Citation Format: Piia Mikkonen, Laura Turunen, Lauri Paasonen, Swapnil Potdar, Lassi Paavolainen, Astrid Murumägi, Olli Kallioniemi, Vilja M. Pietiäinen. Precision cancer medicine based on 3D drug profiling of patient-derived cancer cell spheroid models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5029.

Abstract 991: Development and characterization of patient derived xenografts from central nervous system metastasis reveal minor clone expansion linked with aggressive tumor behavior

Abstract Despite recent advances in cancer therapy, options for central nervous system metastasis (CM) remain limited and patient outcomes are dismal. The lack of tumor models that reflect the heterogeneity and complexity of human CM tumors is a barrier to understanding the underlying biology and to developing novel therapies. Here, we report the development and characterization of 39 patient derived xenograft (PDX) models of CM tumors with histological subtypes representing the disease spectrum. The PDX models successfully grew both in the flank and brains of mice, and retained their histology regardless of the site of implantation. Brain metastasis also formed in 5/12 models after intra-cardiac injection. Multi-omic analysis of the PDX tumor and the patient-matched CM tumor showed that PDXs largely retained the molecular profiles of the tumor from which they were derived, including the retention of aberrations in key driver genes and signaling pathways. PDXs differed primarily from the ungrafted patient tumors by a downregulation in immunity-related pathways, which was expected due to the use of an immuno-compromised host, arguing for the validity of using the flank as a site of implantation. Integrated analysis of mutations and gene expression changes in CM revealed an upregulation of neuronal signaling and DNA damage response, and downregulation of calcium signaling and inflammation. We also assessed the clonal evolution of the PDX from the original patient CM by tracing variant allele frequencies in copy number neutral regions. We found that the PDX tumors were heterogeneous and comprised of multiple tumor clones that were either unique or in common with the original tumor, and displayed evidence of clonal expansion. Two PDXs that underwent phylogeny assessment showed evidence of minor clone expansion. One such PDX was a tumor from a Her2+ breast cancer (CM13), which largely did not molecularly resemble its matched patient tumor but was confirmed to have some shared expressed variants. The PDX was nearly copy number neutral, which included loss of the Her2 amplicon, and had few mutations compared with its parent tumor. Most noteworthy, is that CM13 displayed unique molecular patterns, aggressive growth and was highly metastatic in vivo. From the flank, it led to multiple metastases, including micrometastasis in the brain. This suggests that minor clone takeover may confer tumor aggressiveness and potentiate brain metastasis. The large and diverse repertoire of PDXs developed and characterized in this study provides a new set of tools that will help deepen our understanding of CM and improve preclinical testing for CM therapies. Our data also show that expansion of minor clones could confer aggressive tumor behavior and may be important in tumor evolution in PDX models and human cancers. Citation Format: Ben Yi Tew, Christophe Legendre, Tim Triche, Gerald C. Gooden, Kyle N. Johnson, Rae Anne Martinez, Emanuel F. Petricoin, Mariaelena Pierobon, Joyce O'Shaughnessy, Cindy Osborne, Mark A. Schroeder, Daniel J. Ma, Mark Bernstein, Jann N. Sarkaria, Steven A. Toms, Bodour Salhia. Development and characterization of patient derived xenografts from central nervous system metastasis reveal minor clone expansion linked with aggressive tumor behavior [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 991.

MBRS-56. SYSTEMATIC DRUG REPURPOSING IDENTIFIES DIGOXIN AS A DRUG THAT PROLONGS SURVIVAL IN PDOX MODELS OF GROUPS 3 AND 4 MEDULLOBLASTOMA AT CLINICALLY RELEVANT DOSES

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. While current 5-year EFS is >75 %, recurrent MB continues to have dismal outcomes and survivors face significant treatment-related sequelae. Drug repositioning is a promising strategy which allows for accelerated translation into the clinical setting. To apply this approach to Groups 3 and 4 medulloblastoma, we used a novel algorithm to identify driver signaling pathways by integrating mRNA expression, DNA-copy number, DNA-methylation and DNA-seq profiles from >1,800 patients. By combining these driver pathways with the targeted effects of 1,309 drugs from connectivity map database, we identified the cardiac glycoside family of compounds as potential inhibitors of tumor growth. These findings were first validated in MB-derived cell lines which showed high rates of growth inhibition. Patient-derived orthotopic-xenograft (PDOX) models of Group 3 (ICb-2555MB) and 4 MB (ICb-1078MB) were then treated with digoxin (2 mg/kg i.p.) for 2 cycles of 14 days. Digoxin treatment significantly prolonged survival in both models compared to untreated controls (p=0.001; p=0.031). Studies using the Group 3 model further demonstrated that the observed survival benefit is synergistic with radiation. Furthermore, the average digoxin trough levels in treated mice (2.4 ng/mL +/- 0.4) were similar to target levels in patients taking digoxin, suggesting the potential for rapid translation of these findings into a clinical setting. Finally, digoxin-mediated cell killing occurs in vivo via apoptosis and does not require loss of HIF-1 alpha protein.

Core signaling pathways in ovarian cancer stem cell revealed by integrative analysis of multi-marker genomics data

Tumor recurrence occurs in more than 70% of ovarian cancer patients, and the majority eventually becomes refractory to treatments. Ovarian Cancer Stem Cells (OCSCs) are believed to be responsible for the tumor relapse and drug resistance. Therefore, eliminating ovarian CSCs is important to improve the prognosis of ovarian cancer patients. However, there is a lack of effective drugs to eliminate OCSCs because the core signaling pathways regulating OCSCs remain unclear. Also it is often hard for biologists to identify a few testable targets and infer driver signaling pathways regulating CSCs from a large number of differentially expression genes in an unbiased manner. In this study, we propose a straightforward and integrative analysis to identify potential core signaling pathways of OCSCs by integrating transcriptome data of OCSCs isolated based on two distinctive markers, ALDH and side population, with regulatory network (Transcription Factor (TF) and Target Interactome) and signaling pathways. We first identify the common activated TFs in two OCSC populations integrating the gene expression and TF-target Interactome; and then uncover up-stream signaling cascades regulating the activated TFs. In specific, 22 activated TFs are identified. Through literature search validation, 15 of them have been reported in association with cancer stem cells. Additionally, 10 TFs are found in the KEGG signaling pathways, and their up-stream signaling cascades are extracted, which also provide potential treatment targets. Moreover, 40 FDA approved drugs are identified to target on the up-stream signaling cascades, and 15 of them have been reported in literatures in cancer stem cell treatment. In conclusion, the proposed approach can uncover the activated up-stream signaling, activated TFs and up-regulated target genes that constitute the potential core signaling pathways of ovarian CSC. Also drugs and drug combinations targeting on the core signaling pathways might be able to eliminate OCSCs. The proposed approach can also be applied for identifying potential activated signaling pathways of other types of cancers.

Genomic profiling of intestinal-type sinonasal adenocarcinoma reveals subgroups of patients with distinct clinical outcomes.

Patients with intestinal-type sinonasal adenocarcinoma (ITAC) have an unfavorable prognosis and new therapeutic approaches are needed to improve clinical management. Genetic analysis of 96 ITACs was performed by microarray comparative genomic hybridization and immunohistochemistry and correlated to previously obtained mutation, methylation, and protein expression data, and with pathological characteristics and clinical outcome. Seven copy number alterations (CNAs) were significantly associated with unfavorable clinical outcome: gains at 1q22-23, 3q28-29, 6p22, and 13q31-33, and losses at 4p15-16, 4q32-35, and 10q24. Unsupervised cluster analysis resulted in 5 subgroups of ITAC with significantly distinct genetic signatures and clinical outcomes, independently of disease stage or histological subtype. These data may guide studies to identify driver genes and signaling pathways involved in ITAC. In addition, the subclassification of genetic subgroups of patients with distinct clinical behavior can aid therapeutic decision making and may ultimately lead to personalized therapy with targeted inhibitors.

MTOR co-targeting strategies for head and neck cancer therapy

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. There is an urgent need to develop effective therapeutic approaches to prevent and treat HNSCC. Recent deep sequencing of the HNSCC genomic landscape revealed a multiplicity and diversity of genetic alterations in this malignancy. Although a large variety of specific molecules were found altered in each individual tumor, they all participate in only a handful of driver signaling pathways. Among them, the PI3K/mTOR pathway is the most frequently activated, which plays a central role in cancer initiation and progression. In turn, targeting of mTOR may represent a precision therapeutic approach for HNSCC. Indeed, mTOR inhibition exerts potent anti-tumor activity in HNSCC experimental systems, and mTOR targeting clinical trials show encouraging results. However, advanced HNSCC patients may exhibit unpredictable drug resistance, and the analysis of its molecular basis suggests that co-targeting strategies may provide a more effective option. In addition, although counterintuitive, emerging evidence suggests that mTOR inhibition may enhance the anti-tumor immune response. These new findings raise the possibility that the combination of mTOR inhibitors and immune oncology agents may provide novel precision therapeutic options for HNSCC.