Search

Neurotrophic tyrosine receptor kinase genes (NTRK1/2/3) act as oncogenic drivers across a range of tumors. NTRK fusions occur at low frequency (90% of secretory breast carcinomas. Furthermore, up to 30% of patients (pts) with breast cancer will develop central nervous system (CNS) metastases (mets). Entrectinib is a potent, oral TRKA/B/C, ROS1 and ALK inhibitor, specifically selected for its CNS penetration properties. Entrectinib was evaluated in 3 global phase 1/2 clinical trials (ALKA-372-001 [EudraCT 2012-000148-88], STARTRK-1 [NCT02097810], and STARTRK-2 [NCT02568267]), where it demonstrated strong and durable systemic and intracranial efficacy in pts with NTRK-fp solid tumors, including those with breast cancer. We present updated data from this integrated analysis (data cut-off: 31 October 2018) focusing on pts with breast cancer. The entrectinib trials were conducted at >150 sites in 15 countries, and enrolled pts with locally advanced/metastatic NTRK-fp tumors (with or without baseline CNS mets) confirmed by nucleic acid-based methods. Tumor assessments were performed at the end of cycle 1 (4 weeks) and every 8 weeks thereafter, and evaluated by blinded independent central review (BICR) using RECIST v1.1. Primary endpoints were objective response rate (ORR) and duration of response (DoR) by BICR. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. At clinical cut-off, the overall efficacy-evaluable population included 74 adults from the 3 trials, with 12 different NTRK-fp tumor types and >25 histopathologies. In these pts, ORR by BICR was 63.5% (95% CI 51.5-74.4), including five complete responses (CR) and 42 partial responses (PR). Median (95% CI) DoR, PFS and OS were 12.9 (9.3-not estimable [NE]), 11.2 (8.0-15.7) and 23.9 (16.0-NE) months, respectively. The efficacy-evaluable NTRK-fp breast cancer cohort included 6 pts with a median age of 63 (range 36-67) years; most had an Eastern Cooperative Oncology Group performance status of 0 (3/6; 50%) or 1 (1/6; 17%). Breast cancer tumors were classified as secretory (4/6; 67%; all NTRK3-fp) or non-secretory (2/6; 33%; all NTRK1-fp). Pts had received 0 (3/6; 50%), 1 (1/6; 17%) or ≥4 (2/6; 33%) lines of prior therapy for metastatic disease. At data cut off, the median survival follow-up was 17.4 (range 1.7-23.9) months. ORR was 100% (2 CR, 2 PR; 95% CI 39.8-100.0) in pts with secretory and 50% (1 PR, 1 missing/unevaluable; 95% CI 1.3-98.7) in pts with non-secretory histology. Median (95% CI) DoR, PFS, and OS were 12.9 (4.2-NE), 10.1 (5.1-NE), and 23.9 (5.1-23.9) months, respectively. At baseline, 2 pts had CNS mets per investigator assessment; 1 of these pts had missing response data. CNS mets were confirmed by BICR in the other pt (non-secretory); this pt had received whole brain radiotherapy 2-6 months before starting entrectinib treatment, and had systemic PR and intracranial non-CR/non-progressive disease (non-measurable CNS lesion). The overall integrated safety-evaluable population comprised 504 pts with any gene fusion who received ≥1 dose of entrectinib. Most treatment-related adverse events (TRAEs) were Grade ≤3 (96.1%); the most frequently reported TRAEs were dysgeusia (39.7%) and fatigue (31.5%). Seven breast cancer pts were evaluated for safety, of whom six (85.7%) reported TRAEs; all were Grade ≤3. The most frequently reported TRAEs (each occurring in 3/7 pts; 42.9%) were nausea, anemia, and increased alanine or aspartate aminotransferase. Dose reductions and interruptions due to TRAEs were each reported in 3/7 pts (42.9%); no discontinuations or deaths due to TRAEs were recorded. In this updated integrated analysis, entrectinib induced objective responses in all pts with NTRK-fp breast cancer who had data available, and was generally well tolerated with no discontinuations due to TRAEs. Citation Format: Janice Lu, Collin M. Blakely, Christophe Le Tourneau, Saiama N. Waqar, Todd M. Bauer, Filippo de Braud, David S. Hong, Alexander Spira, Tira Tan, Samuel McCallum, Stuart Osborne, Brian Simmons, Minal Barve. Efficacy and safety of entrectinib in NTRK fusion-positive (NTRK-fp) breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-28.

Background: Somatic alterations in the Epidermal Growth Factor Receptor (EGFR) gene represent a common subset of oncogene-driven lung cancer. Patients who receive EGFR-targeted tyrosine kinase inhibitor (TKI) therapy inevitably develop treatment resistance with limited options after disease progression, with immune checkpoint inhibitor therapy (ICI) typically ineffective. Tumor Immune Microenvironment (TME) characteristics, including T-cell receptor (TCR) sequencing and clonality have been incompletely explored in this population. Methods: We further analyzed our group’s available data from a previous study on therapy-induced evolution of human lung cancer (Maynard et. al, PMID: 32822576). Tumor samples were sorted and subjected to smartseq-based single-cell RNA sequencing. A total of 7495 cells were derived from 17 samples from 10 patients with tumors that harbored EGFR driver gene mutations. This included 1322 gene marker-annotated T-cells. Samples were grouped into three treatment conditions: treatment naïve (TN), residual disease (RD), and progressive disease (PD). We then used the TraCeR tool to infer the TCR α, β, and the complementarity-determining region 3 (CDR3) sequences. Results: T-cells were enriched in RD (55.2%) relative to TN (12.8%) and PD (31.9%) (p 0.255 TN > 0.205 RD) while increased clonal expansion occurred in RD (32 clones from 4 patients) as compared to TN (17 clones from 1 patient) and PD (14 clones from 1 patient) (p Conclusion: T-cell infiltration is greatest in RD, while immunosuppressive genes were significantly expressed in PD. Increased TCR clonal expansion with less clonal diversity in RD suggests the presence of tumor-specific TCR clones and that this may be the optimal time to initiate ICI treatment. Future directions will include the identification of clonally expanded TCR and matched neoantigens to provide potential tailored immunotherapies for patients with TKI-resistant tumors. Citation Format: Turja Chakrabarti, Wei Wu, Daniel L. Kerr, Paul Allegakoen, Trever G. Bivona, Collin M. Blakely. Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 605.

Background Fusions involving the neurotrophic tropomyosin receptor kinase (NTRK) gene family NTRK1, NTRK2, and NTRK3 lead to the expression of chimeric rearrangements in the TRK tyrosine kinase proteins (TRKA, TRKB, and TRKC, respectively) with constitutively active kinase function. NTRK fusions act as oncogenic drivers, and are potential therapeutic targets across a broad range of tumor types including breast cancer. Entrectinib is a CNS-active, oral, potent inhibitor of TRKA/B/C, ALK and ROS1 designed to cross the blood-brain-barrier. Here we present integrated efficacy data from three trials of entrectinib in NTRK fusion-positive solid tumors focusing on a small cohort of patients with breast cancer, and safety data from the integrated safety population. Methods Patients with locally advanced/metastatic NTRK fusion-positive tumors (with or without baseline CNS disease) confirmed by nucleic acid-based methods were enrolled in three global phase 1/2 entrectinib trials at >150 sites in 15 countries (ALKA-372-001 [EudraCT 2012-000148-88], STARTRK-1 [NCT02097810], STARTRK-2 [NCT02568267]). Disease burden was assessed per blinded independent central review (BICR) using RECIST v1.1 after cycle 1 (4 weeks) then every 8 weeks thereafter. The primary endpoints were objective response rate (ORR) and duration of response (DOR) by BICR. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. Results The total efficacy-evaluable population comprised 54 adult patients with advanced/metastatic NTRK fusion-positive tumors; within this population 10 different tumor types and >19 histopathologies were identified. In the overall integrated analysis population, responses to treatment with entrectinib occurred in all tumor types and included 4 complete responses (7.4%). The ORR by BICR was 57.4% (95% CI 43.2-70.8), and median (95% CI) DOR, PFS, and OS were 10.4 (7.1-not estimable [NE]), 11.2 (8.0-14.9), and 20.9 (14.9-NE) months, respectively. In the cohort of 6 patients with NTRK fusion-positive breast cancer, the median age was 63 years (range 36-67 years). Three patients reported prior systemic therapies (6, 4 and 1 prior therapies). The majority of patients had an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1 (66.7%). Two patients had investigator-assessed CNS metastases at baseline; 1 patient (16.7%) had previously received radiotherapy in the brain 2-6 months prior to treatment with entrectinib. ORR by BICR was 83.3% (95% CI 35.9-99.6); 2 were complete responses, 3 were partial responses, and 1 patient had missing/unevaluable data. Median (95% CI) DOR, PFS, and OS were: 12.9 (4.2- NE), 10.1 (5.1-NE), and NE (5.1-NE) months, respectively. The integrated safety population comprised 355 patients who received ≥1 dose of entrectinib, of which 60.5% of patients had grade 1 or 2 treatment-related adverse events (TRAEs), 27.6% had grade 3, 3.4% had grade 4, and there were no grade 5 TRAEs. The most frequently reported TRAEs were dysgeusia (41.4%), fatigue (27.9%), dizziness (25.4%) and constipation (23.7%). TRAEs led to dose reductions in 27.3%, interruptions in 25.4% and discontinuations in 3.9% of patients. Conclusion In this integrated analysis of global multicenter clinical trials, entrectinib was well tolerated and induced clinically meaningful, durable responses in patients with NTRK fusion-positive breast cancer. Study Sponsor: Ignyta, Inc., a wholly owned subsidiary of F. Hoffmann-La Roche Ltd. Citation Format: Collin Blakely, Christophe Le Tourneau, Janice Lu, Saiama N. Waqar, Xinhui Huang, Bann-mo Day, Brian Simmons, Minal Barve. Entrectinib in NTRK fusion-positive breast cancer: Integrated analysis of patients enrolled in STARTRK-2, STARTRK-1 and ALKA-372-001 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-11.

Introduction: Single-cell RNA sequencing of dissociated tumors enables the profiling of cellular states in fine detail, but erases the cellular organization of the analyzed tissue. Spatially resolved transcriptomics (SRT) using 10X Genomics’ Visium platform combines histological staining and RNA sequencing by capturing each transcript across spatially barcoded microarrays. SRT yields gene-expression matrices resolved within 55-micron array spots, and creates opportunities to explore how lung biology is affected by lung adenocarcinoma and how tumor cells and the proximal microenvironment are modulated by targeted therapy. Methods: SRT reactions (n=8) were performed on surgical specimens from human lung adenocarcinomas driven by kinase domain mutations of EGFR (exon 19 deletion, L858R point mutation, exon 20 insertion). SRT reactions analyzed primary lung cancer (n=5) or paired tumor-adjacent lung tissues (n=3). Results: 28458 array spots were recorded across all samples, and array spots captured a median of 5800 total transcripts and a median of 2416 unique transcripts. Single marker gene expression and unsupervised clustering across array spots corresponded with histological annotations of lung structures and adenocarcinoma. Integration of single-cell RNA sequencing data from lung adenocarcinoma specimens permitted mapping and quantification of 15 major cell types, including cancer cells, T- and B- lymphocytes, macrophages, dendritic cells, fibroblasts, and endothelial cells. Compared with paired tumor-adjacent lung tissues, adenocarcinoma tissues contained fibroblast-enriched desmoplasia and B-cell enriched tertiary lymphoid structures. In a clinical case with resistance to Osimertinib, the standard tyrosine kinase inhibitor, gene signature analysis of cancer-containing array spots revealed enrichment of gap-junction, fatty acid metabolism, and kynurenine pathway signatures compared to treatment naïve array spots. Conclusion: This pilot study demonstrates the feasibility of using SRT in lung adenocarcinoma. Paired with a single-cell atlas of lung adenocarcinoma during targeted therapy, this approach enables hypothesis-generation to investigate the alterations of tumor and tumor microenvironmental architecture in relation to targeted therapy. Citation Format: Daniel L. Kerr, Wei Wu, Whitney Tamaki, Anatoly Urisman, Yu-Ting Chou, Philippe Gui, David M. Jablons, Trever G. Bivona, Collin M. Blakely. Spatially resolved transcriptomics of cellular architecture in EGFR-mutated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3808.

Introduction: Increasing our understanding of drivers of mutagenesis in lung cancer is critical in our efforts to prevent tumor reoccurrence and resistance. Results: Using the multi-region TRACERx lung cancer study, we uncovered that APOBEC3B is significantly upregulated when compared with other APOBEC family members in EGFR driven lung cancer and identified subclonal enrichment of APOBEC mutational signatures. To model APOBEC mutagenesis in lung cancer, several novel EGFR mutant mouse models containing a human APOBEC3B transgene were generated. Using these models, it was uncovered that APOBEC3B expression is detrimental at tumor initiation when expressed continuously in a p53 wildtype background. This detrimental effect is likely due to elevated chromosomal instability, which was observed to increase significantly with APOBEC3B expression in an EGFR mutant TP53 deficient mouse model. Induction of subclonal expression of APOBEC3B in an EGFR mutant mouse model with tyrosine kinase inhibitor (TKI) therapy resulted in a significant increase in resistant tumor development. Significant downregulation of the base excision repair gene uracil-DNA glycosylase (UNG) was also observed in APOBEC3B expressing mice, which paralleled findings in patient tumors and cell lines treated with TKI therapy. Finally, a mouse mutational signature was identified in APOBEC3B expressing cell lines, reinforcing the idea that APOBEC driven mutagenesis contributes to TKI resistance. Conclusion: This study demonstrates a unique principle by which targeted therapy induces changes within tumors ideal for APOBEC driven tumor evolution, fueling therapy resistance. Citation Format: Manasi Mayekar, Deborah Caswell, Natalie Vokes, Emily K. Law, Wei Wu, William Hill, Eva Gronroos, Andrew Rowan, Maise Al Bakir, Clare Weeden, Caroline E. McCoach, Collin M. Blakely, Nuri Alpay Temiz, Ai Nagano, Daniel L. Kerr, Julia K. Rotow, Oriol Pich, Franziska Haderk, Michelle Dietzen, Carlos Martinez Ruiz, Bruna Almeida, Lauren Cech, Beatrice Gini, Joanna Przewrocka, Chris Moore, Miguel Murillo, Bjorn Bakker, Brandon Rule, Cameron Durfee, Shigeki Nanj, Lisa Tan, Lindsay K. Larson, Prokopios P. Argyris, William L. Brown, Johnny Yu, Carlos Gomez, Philippe Gui, Rachel I. Vogel, Elizabeth A. Yu, Nicholas J. Thomas, Subramanian Venkatesan, Sebastijan Hobor, Su Kit Chew, Nicholas McGranahan, Nnennaya Kanu, Eliezer M. Van Allen, Julian Downward, Reuben S. Harris, Trever Bivona, Charles Swanton. Targeted cancer therapy induces APOBEC fueling the evolution of drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2197.

Introduction: Lung cancer is the leading cause of cancer-related mortality, with non-small cell lung cancer (NSCLC) as the most common form. Targeting the immune system with immune checkpoint inhibitors has transformed NSCLC treatment, highlighting the crucial contribution of immune cells and the tumor microenvironment (TME) to NSCLC initiation and progression. However, oncogenic-driven cancers treated with targeted molecular therapies have not benefited from immunotherapy, suggesting that other mechanisms of disease progression and resistance are involved. Macrophages in the lung TME are associated with disease outcomes but their exact role in tumor progression and resistance is less well understood. Characterizing their function using high-resolution methods, such as single cell RNA sequencing (scRNAseq), has the potential to identify mechanisms by which macrophages promote lung cancer progression and drug resistance. Methods: We performed scRNAseq analyses on individual human NSCLC tumor biopsies collected at different treatment states. Samples were obtained from patients before initiating systemic targeted therapy (treatment-naïve, TN), at the residual disease state, which includes samples taken at any time during treatment with targeted therapy while the tumor was regressing or at a stable state by standard clinical imaging (residual disease, RD), and upon the subsequent and clear progressive disease, as determined by standard clinical imaging, at which point the tumors had establishment of acquired drug resistance (progressive disease, PD). Samples were primarily EGFR-, BRAF-, and ALK-driven tumors. Macrophages from lung tumor biopsies were identified and annotated using SingleR, which is a computational method for unbiased cell type recognition of scRNAseq, and then characterized. Results: We identified 678 macrophages that clustered into 7 distinct groups (Figure 1A). Clusters MF0, MF1, MF3, and MF5 had significant differences across treatment time points. Cluster MF0 had significantly less RD cells and was characterized by expression of genes associated with neutrophil and leukocyte recruitment. Cluster MF5 had significantly more PD cells and was characterized by expression of genes associated with T-cell activation (Figures 1B and 1C). Conclusion: We annotated macrophages in the lung TME using scRNAseq and characterized macrophage changes across different treatment states. Our findings suggest there are less macrophages recruiting neutrophils and leukocytes in RD compared to TN and PD, and more T-cell activating macrophages in PD compared to TN and RD, which could have potential therapeutic and prognostic implications. Citation Format: Elizabeth A. Yu, Wei Wu, Philippe Gui, Caroline E. McCoach, Collin M. Blakely, Stephanie A. Christenson, Trever G. Bivona. Deciphering macrophage function in lung tumor microenvironment and disease progression [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 2753.

Background: Patients (pts) with EGFRm T790M-negative NSCLC with progressive disease (PD) on an EGFR TKI have limited therapy options. CD73 may promote immune evasion and is overexpressed in EGFRm NSCLC, suggesting the potential for combining CD73 blockers with EGFR TKIs. Oleclumab (MEDI9447) disrupts immune evasion by binding to CD73. This phase 1b/2, multicenter, dose escalation/expansion study (NCT03381274) evaluates the safety and efficacy of oleclumab plus osimertinib (osi) in locally advanced/metastatic EGFRm NSCLC. Methods: Pts had histologically or cytologically confirmed T790M-negative EGFRm NSCLC by local testing, a TKI-sensitive EGFR mutation (exon 19 del, L858R), ECOG PS 0-1, PD on a 1st or 2nd generation TKI, and no prior osi. Oleclumab 1500 mg (DL1) or 3000 mg (recommended Phase 2 dose, RP2D) IV Q2W + osi 80 mg PO QD were given until PD or intolerable AEs. Primary endpoints included AEs, SAEs and ORR by RECIST v1.1. DLTs were predefined and reviewed by a dose-escalation committee. Secondary endpoints included duration of response, disease control rate, PFS and OS. Results: As of November 9, 2020, 5 pts received DL1 and 21 pts received RP2D. Minimum follow-up was 44 wks. For the 5 pts receiving DL1, safety profile and response rate were generally similar to RP2D and no DLTs occurred. All of the 21 pts receiving RP2D had ≥1 treatment-emergent AE (TEAE), 38.1% had Grade 3/4 TEAEs and 23.8% had SAEs. Treatment-related AEs (TRAEs) occurred in 81.0% (19.0% Grade 3/4); none were SAEs or deaths. The most common TRAEs were rash (33.3%), stomatitis (28.6%), diarrhea (23.8%) and paronychia (23.8%). At data cutoff, 38.1% remained on treatment; 47.6% discontinued due to PD, with death, pt decision and AE (pneumonitis) as other causes. Response and survival for pts receiving RP2D are shown in Table 1. Conclusions: Oleclumab + osi was well tolerated at RP2D. Compared to T790M-negative EGFRm NSCLC pts in a prior study of osi monotherapy, ORR was similar (21% vs 19%) but mPFS was longer (2.8 vs 11.0 mos). Table 1.Disease response and survivalOutcomeN=21Best overall response, n (%)CR0PR4 (19.0)SD13 (61.9)PD3 (14.3)Not evaluable1 (4.8)ORR, n (%)4 (19)Median duration of response, months (range)Not assessable (9.2-11.1)Disease control rate, n (%)17 (81.0)Median PFS (95% CI), months11 (3.7-not evaluable)6-month PFS rate (95% CI), %63.5 (38.3, 80.7)9-month PFS rate (95% CI), %52.9 (29.0, 72.1)Median OS (95% CI), monthsNot reached (11.7-not evaluable)6-month OS rate (95% CI), %100 (100, 100)9-month OS rate (95%CI), %90.0 (65.6, 97.4) Citation Format: Dong-Wan Kim, Sang-We Kim, D. Ross Camidge, Naiyer A. Rizvi, Kristen A. Marrone, Xiuning Le, Collin Blakely, Keunchil Park, Gee-Chen Chang, Sandip Pravin Patel, Zachary A. Cooper, Rakesh Kumar, Ramin Samadani, Rebecca McCombs, Michael Pluta, Kevin Yufeng Wu, Suresh Ramalingam. CD73 inhibitor oleclumab plus osimertinib for advanced EGFRm NSCLC: First report of a Phase 1b/2 study [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 CT163.

Despite multimodal therapy and ever-improving surgical technique, glioblastoma (GBM) maintains its poor prognosis due to the chemotherapeutic resistance it builds over time, eventually rendering existing agents ineffective. A well-characterized phenomenon that takes place particularly in mesenchymal GBM tissue is the Warburg Effect, characterized by a shift from oxidative phosphorylation to glycolysis as a primary means to derive energy. Pyruvate Dehydrogenase Kinase 1 (PDK1) is one upregulated molecule in this shift toward anaerobic metabolism that is known for phosphorylating pyruvate dehydrogenase, the “gate keeper” of the TCA cycle. We sought to characterize further the role of PDK1 in terms of its metabolic role and any extra-metabolic functions it may serve. By silencing PDK1 and considering changes in downstream expression, we hope to understand what other factors serve as contributors to metabolic alterations seen in GBM. Elucidating the mechanism of this crucial shift more thoroughly, especially without the prominently cited role of PDK1, can begin to expose new targets for interrupting tumor maintenance and proliferation by affecting its metabolism. In this study we used differential expression of two vectors in which PDK1 was silenced, shPDK1 and SCBT-PDK1. GSEA analysis was done and analyzed to identify classes of genes or proteins that are over-represented and to identify significantly enriched or depleted groups of targets whose expression was vastly changed by silencing of PDK1. Using GSEA heat map data, gene targets whose expression was noted to be consistently changed with silencing of PKD1 targets were identified. Datamining and immunoblot analyses were then conducted to evaluate for expression of these genes in correlating GBM specimens. Ultimately, we further characterize PDK1’s global role in GBM and evaluate non-metabolic effects of PDK1 that may still affect survival and proliferation of GBM tumors. Citation Format: Neha Jain, Maheedhara Guda, Collin Labak, Chase Smith, Simon Park, Andrew Tsung, Kiran Velpula. The role of PDK1 in metabolic reprogramming of mesenchymal glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1848.

Glioblastoma (GBM) is an aggressive diffuse glioma with poor prognosis due to lack of sound therapeutic options. As in many aggressive tumors, GBMs create a hypoxic microenvironment and switch to glycolytic metabolism from oxidative phosphorylation in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting influx of glucose via targeting GLUT1 may reverse the Warburg Effect and selectively slow tumor progression. Nanostring data suggests an aberrant metabolic clincial profile in our cohort of GBM patients. Datamining studies and immunoblot analysis were conducted on these same human GBM specimens to demonstrate upregulated GLUT1. Mass spectrometric analysis was performed on GLUT1 precipitated from mesenchymal subtype patient-derived xenograft (PDX). Zonula Occludens adhesion studies were carried out to determine GLUT1 inhibition's effects on cell-cell adhesion structures. Finally, ATP, lactate release, and glucose uptake assays were performed on PDX and GLUT1-silenced PDX to demonstrate a normalized metabolic phenotype when GLUT1 is silenced. Our work suggests that GLUT1 overexpression plays a major role in the metabolic profile of mesenchymal subtype GBM, and that targeting its overexpression could prove effective in reversing the Warburg Effect in GBM cells and ultimately improving patient outcomes. Citation Format: Collin M. Labak, Maheedhara R. Guda, Neha Jain, Chase P. Smith, Charles P. Cain, Andrew J. Tsung, Kiran K. Velpula. GLUT1 as a metabolic target in glioblastoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3594.

Background Gene fusions involving NTRK (neurotrophic receptor tyrosine kinase) genes result in the expression of chimeric TRK proteins with uncontrolled kinase activity, leading to oncogenic transformation and unrestrained cancer cell proliferation, which confers oncogenic potential across many histopathologies. Entrectinib is a central nervous system (CNS)-active, potent inhibitor of all TRK kinases, as well as ROS1. We present integrated efficacy and safety data for entrectinib in patients with locally advanced/metastatic NTRK fusion-positive (NTRK-FP) solid tumors enrolled in global (>150 sites, 15 countries) Phase I/II entrectinib trials (ALKA-372-001 [EudraCT 2012-000148-88], STARTRK-1 [NCT02097810], STARTRK-2 [NCT02568267]), focusing on a cohort of patients with NSCLC. Methods The analysis included patients with TRK inhibitor-naïve solid tumors (with or without baseline CNS disease) harboring an NTRK fusion identified via nucleic acid-based diagnostic platforms. Tumor assessments were performed at week 4 and every 8 weeks thereafter. Disease burden was assessed per blinded independent central review (BICR) by RECIST v1.1. Primary endpoints (per BICR): overall response rate (ORR) and duration of response (DOR). Key secondary endpoints: progression-free survival (PFS), overall survival (OS) and safety Results In the total efficacy-evaluable population (n=54 adult patients with advanced/metastatic NTRK-FP solid tumors, median age 57.5 years, 59.3% female, 22.2% baseline CNS disease, >19 histopathologies across 10 cancer types), ORR (BICR) was 57.4% (95% CI 43.2-70.8), with 4 complete responses (7.4%); responses were seen in all tumor types. Median DOR: 10.4 months (95% CI 7.1-NR); median PFS: 11.2 months (95% CI 8.0-14.9); median OS: 20.9 months (95% CI 14.9-NR). In the cohort of patients with NTRK-FP NSCLC (n=10), ORR (BICR) was 70.0% (7/10; 7/7 adenocarcinoma NSCLC, 0/3 squamous cell or unclassified/undifferentiated NSCLC). In NSCLC patients with investigator-assessed baseline CNS disease (n=6), 4/6 had an intracranial response (2 complete, 2 partial); 1 had stable disease, and 1 was not evaluable. In the safety population (68 patients with NTRK-FP solid tumors who received at least 1 dose of entrectinib) most treatment-related adverse events (AEs) were grade 1-2; grade 3: 32.4% of patients, grade 4: 2.9% of patients; no grade 5 treatment-related AEs. AEs resulted in dose reduction in 39.7% of patients and discontinuation in 4.4% of patients. Conclusion In this integrated analysis of global multicenter clinical trials, entrectinib was well tolerated and induced clinically meaningful, durable systemic and CNS responses in patients with NTRK-FP solid tumors, including those with NSCLC. Citation Format: Robert Doebele, Luis Paz-Ares, Anna F. Farago, Stephen V. Liu, Sant P. Chawla, Diego Tosi, Collin M. Blakely, John C. Krauss, Darren Sigal, Lyudmila Bazhenova, Tom John, Benjamin Besse, Jürgen Wolf, Takashi Seto, Edna Chow-Maneval, Chenglin Ye, Brian Simmons, George D. Demetri. Entrectinib in NTRK-fusion positive (NTRK-FP) non-small cell lung cancer (NSCLC): Integrated analysis of patients enrolled in three trials (STARTRK-2, STARTRK-1 and ALKA-372-001) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT131.

Background: EGFR tyrosine kinase inhibitors are effective for treatment of non-small cell lung cancer. Benefit from these therapies is often transient due to residual disease leading to acquired resistance. Osimertinib now used in first-line setting, but mechanisms of resistance are ill-defined and no effective second-line therapies exist. Disease progression occurs through tumor evolution that involves the emergence of distinct genetic alterations and non-genetic changes in cell state. In this study, we determine the molecular drivers of drug sensitivity and tumor evolution which forms the basis of clinical genomic heterogeneity. We show that the increase in genomic instability correlates with increased chromosomal alterations or rate of mutation which creates evolutionary dependencies. Method: Using our previously established EGFR-TKI acquired resistance models, we observed heightened TPX2/AURKA activity. We measured the temporal activation of TPX2/AURKA in sensitive phase, a drug-tolerant phase (residual phase) and acquired resistant phase. Chromosomal segregation errors, mitotic deformation and aneuploidy were used as a functional read-out to the measure genomic instability upon EGFR-TKI treatment. Using clinical specimens, the contribution of non-genetic TPX2/AURKA axis in disease progression was evaluated. Results: In-vitro models of residual disease exhibited increased AURKA activation via upregulation of its co-activator TPX2. Pharmacological inhibition of AURKA activation by MLN8237 together with osimertinib enhanced the magnitude of response and forestalled the emergence of resistance. Therefore, tumor cells reply on AURKA to transition from a sensitive to drug-tolerant phase and is maintained in acquired resistance. Since high levels of TPX2 and AURKA are known to cause mitotic errors and polyploidy, we surveyed for mitotic defects induced by EGFR-TKI treatment. EGFR-TKI resulted in a marked accumulation of errors in centrosome biogenesis, spindle assembly, and chromosome segregation resulting in polyploid cells with abnormal DNA content indicating persistent mitotic stress is a feature of EGFR inhibition. We conclude that the abnormal activation of TPX2/AURKA leaves a signature of defects associated with mitotic stress, a prominent feature of genomic instability. Examination of clinical samples with T790M mutation or MET amplification upon disease progression had high levels of TPX2. This nominates TPX2 as a novel biomarker therapeutic resistance in a significant fraction of EGFR-mutant lung cancers. Conclusion: Our results indicate that heightened TPX2/AURKA activation is required for tumor cells to transition out of sensitive cell state and persist upon drug pressure. This deregulation creates a highly genomically unstable environment and give rise to genomic heterogeneity providing a fertile ground for the subsequent survival of fittest subclones. Citation Format: Khyati N. Shah, Roma Bhatt, Julia Rotow, Julia Rohrberg, Victor Olivas, Victoria E. Wang, Jonathan Kuhn, Sophie Dumont, Frank Mccormick, Andrei Goga, Collin M. Blakely, Trever G. Bivona, Sourav Bandyopadhyay. Non-genetic TPX2/AURKA signaling facilitates tumor evolution in EGFR-TKI resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2902.

With the advent of precision medicine, EGFR inhibitors are a major breakthrough in the treatment of EGFR-mutant non-small cell lung cancer (NSCLC). Although these EGFR-TKI therapies often elicit profound initial therapeutic responses, their effects are transient due to residual disease. This residual disease and subsequent disease progression occurs through tumor evolution and molecular drivers behind the formation, maintenance and evolution of residual disease and acquired resistance have remained elusive. Although in many cases, pre-existing clones with bona fide genetic resistance have been identified, majority of patients have undetectable resistance causing genetic alterations, suggesting that nongenetic alterations may drive altered cell state and signaling associated with EGFR inhibitor resistance. Using EGFR mutant lung cancer cells, we developed several in vitro models of acquired resistance to third-generation EGFR-TKI inhibitors, osimertinib and rociletinib, and a chemical screen revealed that Aurora kinase inhibitors are highly synergistic when combined with third-generation EGFR inhibitors. Resistant cells harbored high activation of AURKA mediated by upregulation of its co-activator protein TPX2. In in vitro and in vivo models of acquired resistance, the combination induced potent cell death by reactivating BIM-mediated apoptosis. We found that tumors from patients progressing on first- and third-generation EGFR TKIs often harbored high levels of TPX2, indicating that AURKA is likely activated and driving resistance in a significant fraction of EGFR-mutant lung cancers. By tracking the kinetics of AURKA activation, we tested if AURKA activity is required for the formation and maintenance of residual cells leading to acquired resistance. In our sensitive in vitro models, either single-agent EGFR-TKI, MLN8237 or the combination enhanced the magnitude of response and forestalled the emergence of resistance as compared to monotherapies. We tested the combination in an EGFR L858R patient-derived xenograft (PDX) tumor model generated from a residual disease surgical specimen. In this PDX the combination robustly impaired tumor growth compared to both single agents alone and in most cases induced tumor regression This novel synthetic lethal interaction between EGFR TKIs and Aurora kinase inhibitors has important implications for the development of new treatment strategies for third-generation EGFR-TKI. These results invite a paradigm aimed at preventing the emergence of resistance. Citation Format: Khyati N. Shah, Roma Bhatt, Julia Rotow, Julia Rohrberg, Victor Olivas, Golzar Golzar Hemmati, Gregor Krings, Henry J. Haringsma, Andrew D. Simmons, Thomas C. Harding, Andrei Goga, Collin Blakely, Trever Bivona, Sourav Bandyopadhyay. Aurora kinase A drives the evolution of resistance to third-generation EGFR inhibitors in lung cancer [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 1957.

Glioblastoma (GBM) is the most aggressive brain cancer, afflicting nearly 1 in 6 adult patients with brain tumors. Currently, no effective curative treatment for this CNS neoplasm exists. The high prevalence and dismal prognosis of GBM increases the need to identify novel therapeutic targets. Previous research has suggested that increased expression of focal adhesion kinase (FAK) may play a role in tumor metabolism. Our preliminary data using datamining approach of the TCGA and Oncomine™ databases showed that mRNA levels of FAK correlate with expression levels of the glucose transporter GLUT1. Subsequent mass-spectrometric and immunoprecipitation experiments provided evidence that FAK binds to GLUT1, while immunohistochemistry and immunocytochemistry experiments confirmed their interaction. Western blot analysis using shRNA to silence FAK demonstrated a significant decrease in GLUT1 expression. Cells treated with the same FAK shRNA showed decreased metabolites from anaerobic glycolysis, instead relying on oxygen-dependent metabolism utilizing oxidative phosphorylation for their energy supply. Collectively, we propose that FAK is a viable target in modulating glucose uptake and metabolism in GBM. Citation Format: Kiran Kumar Velpula, Michael Pajor, Maheedhara R. Guda, Collin M. Labak, Simon Park, Andrew J. Tsung. FAK overexpression upregulates glucose metabolism in glioblastoma [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 1455.

Glioblastoma (GBM) is the most common form of adult primary malignant brain tumor with a poor prognosis. Despite advances in standard treatments, such as surgery, radiation, and adjuvant chemotherapy, outcome remains poor. To further develop effective therapeutic approaches, it is essential to garner a better understanding of GBM progression. Substantial research has shown that GBM's progression is associated with metabolic remodeling, that includes the use of aerobic glycolysis as the main source of energy. Here, we report the novel role of Mitofusin 1 (MFN1), a key regulator of mitochondrial outer membrane fusion in glycolysis. We first established that MFN1 is highly expressed in GBM when compared to its isoform MFN2 using TCGA (The Cancer Genome Atlas) and OncomineTM glioma datasets. Silencing MFN1 using MFN1-shRNA in GBM10 cells showed decreased expression levels of PDK1 and HIF1-alpha as observed in western blotting and RT-PCR analysis. Interestingly, c-myc expression was significantly reduced in the shMFN1 treated GBM 10 cell. In addition, RNASeq analysis conducted on shPDK1 treated GBM-10 cells revealed reduced MFN1 levels. Further silencing of MFN1 showed increased OCR and decreased ECAR using seahorse XFp bio analyzer. Collectively, our preliminary data points to MFN1 as a candidate metabolic oncogene in GBM, and targeting MFN1 will be a novel GBM treatment. Citation Format: Maheedhara Reddy Guda, Swapna Asuthkar, Collin M. Labak, Chase P. Smith, Andrew J. Tsung, Kiran Velpula. Targeting deregulated expression and function of Mitofusin 1 in glioblastoma [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 5494.

Medulloblastoma (MB) is a pediatric brain tumor comprising of four molecular subgroups, with Group 3 and 4 tumors characterized by particularly poor prognosis. While research has advanced our understanding of MB, there is still a pressing need for new knowledge of the functional processes underlying its progression due to a scarcity of therapeutics. Recent studies have identified Orthodenticle homeobox 2 (Otx2), a cerebellar transcription factor, to be highly expressed in MB Group 3/4 subtypes. Datamining studies from Northcott and Robinson datasets revealed co-expression of Otx2 and c-Myc in Group 3/4 tumors. DAB staining conducted in human MB specimens showed increased expression of Otx2 and c-Myc. Colocalization studies confirmed datamining results. Next, we used a clinical Otx2 inhibitor, all-trans retinoic acid (ATRA), in a PDX cell line, P9, and in UW228 cells. Western blot analysis demonstrated reduced levels of c-Myc along with Otx2. In another experiment, we used the c-Myc/Max inhibitor 10058-F4 and observed comparable results to Otx2 inhibition. Real time PCR and immunocytochemistry experiments confirmed the aforementioned results. Further, treatment with ATRA and 10058-F4 showed reduced proliferation and migratory abilities in p9 and UW228 when compared to their respective controls. Mass spectrometry conducted on immune-precipitated Otx2 in P9 cells revealed a plethora of novel interaction partners that drive tumor growth and progression. In addition, western blot analysis in ATRA treated cells showed reduced expression of Max, suggestive of the existence of a feedback loop that link with Otx2 and c-Myc. Collectively our studies highlight Otx2 as a potential contributor to the malignant phenotype and describe its role in mediating the aggressiveness of Group 3/4 medulloblastoma. Citation Format: Yining Lu, Maheedhara R. Guda, Swapna Asuthkar, Ian Purvis, Collin M. Labak, Neha Jain, David J. Daniels, Sarah E. Martin, Andrew J. Tsung, Kiran K. Velpula. Otx2 and c-Myc association drives tumor progression in medulloblastoma molecular subtypes 3/4 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3537. doi:10.1158/1538-7445.AM2017-3537

Glioblastoma multiforme (GBM) remains one of the most lethal and difficult-to-treat cancers of the central nervous system despite the existence of multi-modal therapeutic approaches. The poor prognosis in GBM patients is due in part to its resistance to available treatments, which calls for identifying novel molecular therapeutic targets. In this study, we identified an interesting biomarker, Hairy/Enhancer-Of-Split related with YRPW Motif (HEY1), a mediator of Notch signaling, which contributes to the pathogenesis of GBM. Datamining studies and our immunohistochemistry and immunoblot analysis showed that HEY1 is highly expressed in GBM patient specimens. Recent studies indicate that methylation status may control the expression of cancer phenotype. Our studies using bisulphite sequencing on the patient samples showed that HEY1 promoter was hypermethylated in the normal brain when compared to GBM specimens. Treatment on the 4910 and 5310 xenograft cell lines with sodium butyrate (NaB) greatly decreased HEY1 expression owing to its promoter hypermethylation with a concomitant increase in DNMT1 expression confirming that promoter methylation regulate the HEY1 expression in GBM. NaB treatment also induced cell apoptosis as evaluated by flow cytometric analysis. Silencing HEY1 reduced invasion, migration and proliferation in 4910 and 5310 cells. Further, immunoblot and q-PCR analysis demonstrated the existence of a potential positive regulatory loop between HEY1 and p53. Additionally, DNA microarrays with HEY1 recombinant protein demonstrated good correlation with p53 and provided various bonafide targets of HEY1. Collectively, these studies suggest HEY1 as an important predictive marker for GBM, thus indicating a potential target for future GBM therapy. Citation Format: Maheedhara Reddy Guda, Andrew J. Tsung, Swapna Asuthkar, Collin M. Labak, Yining Lu, Sarah E. Martin, Prasad V. Durbaka, Kiran K. Velpula. Methylation regulates HEY1 expression in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3535. doi:10.1158/1538-7445.AM2017-3535

Treatment of prostate cancer is still clinically challenging due to lack of reliable markers for diagnosis and prognosis. Serum prostate specific antigen (PSA) is of limited clinical utility due to poor sensitivity and specificity. A large number of false positives (50%) with traditional PSA testing lead many patients to undergoing unnecessary prostate biopsy and exposure to unnecessary risk of complications. So the goal of this study is to identify a reliable, non-invasive biomarker that can distinguish patients with benign, indolent and aggressive prostate cancer in various clinical settings. MicroRNAs (miRNAs) are small noncoding (18 to 28 nucleotides) RNA molecules that are present in all human cells, and their expression patterns are correlated with many cancer types, including CaP. In our results, we found a significant differential expression (p=0.013) of miR-301a in both tumor tissues (Gleason-6 and -7) and serum samples in comparison to benign prostatic hyperplasia (BPH) or adjacent benign samples. We observed a negative correlation between miR-301a and RUNX3 expression in human CaP samples suggesting tumor suppressive role RUNX3 might be compromised in CaP. To determine MiR-301a regulation on RUNX3, we evaluated miR-301a and RUNX3 protein in panel of prostate cancer cell lines, normal prostate epithelial cells and Benign Prostate Hyperplasia (BPH). Similar results such as inverse correlation between miR-301a and RUNX were found in cell culture models. Also, over expression of miR-301a down regulates RUNX3 activation in prostate cancer cell lines and silencing miR-301a expression reverts RUNX3 activation in BPH cells. While evaluating the reporter activity of RUNX3 either by co-transfecting with mimic or inhibitor of miR-301a in BPH and prostate cancer cells: over expression of miR-301a down regulated RUNX3 reporter activity which corresponded with RUNX3 protein expression. Similarly, silencing miR-301a reverted RUNX3 promoter activity and protein expression suggesting RUNX3 is a direct target of miR-301a in CaP cells. Silencing miR-301a reverted the pro-apoptotic function of RUNX3, results in reduced colony formation, adhesion, invasion and migration of CaP cells. Investigating the mechanistic link miR-301a and RUNX3 may explore whether it could facilitate clinical decision making such as the decision to proceed with early surgery rather than active surveillance for intermediate risk prostate cancer patients deemed to be at high risk of progression. Citation Format: Venkatesh Kolluru, Balaji Chandrasekhar, Collin McKenzie, Houda Alatassi, Murali Ankem, Chendil Damodaran. Inhibition of tumor suppressor function of RUNX3 by miR-301a facilitates the progression of castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5445. doi:10.1158/1538-7445.AM2017-5445

Glioblastoma is an aggressive, high-grade tumor with poor prognosis due to lack of sound therapeutic options. Mesenchymal subtype GBMs are particularly difficult to treat because they tend to proliferate in the sub ventricular zone, an area that breeds stem-like neural cells and proves nearly impossible to access for surgical resection. They also contribute to creating a hypoxic microenvironment and switch to glycolytic metabolism in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting the glucose transporter, GLUT1, through cytoskeletal components that traffic it to the cell membrane may reverse the Warburg Effect, attenuating further tumorigenesis and decreasing the stemness of such cells. Datamining studies and immunoblot analysis conducted on human glioblastoma patient specimens (hGBM) demonstrated that GLUT1 is highly upregulated. Limiting dilution assay conducted using GLUT1 inhibitors- fasentin and 2-Deoxy-D-glucose reduced the proliferation in mesenchymal cancer stem cell (CSC) subtype in in vitro culture. Simultaneously, mass spectrometric analysis revealed significant association between GLUT1 and β-tubulin 4 (TUBB4) in mesenchymal subtyped cells. This association was further confirmed by large-sample datamining and in immunoprecipitation studies from hGBM specimens, suggesting that TUBB4 may be a viable target in deterring the trafficking of upregulated GLUT1 to the membrane. Collectively, these studies confirm that GLUT1 is associated with TUBB4, and that targeting TUBB4 via siRNA or colchicine derivatives could prove effective in reversing the Warburg Effect in GBM cells and ultimately improve patient outcomes. Citation Format: Collin M. Labak, Maheedhara R. Guda, Swapna Asuthkar, Neha Jain, Yining Lu, Ian Purvis, Jack Tuszynski, Ichiro Nakano, Andrew J. Tsung, Kiran K. Velpula. β-Tubulin inhibitors reduce GLUT1 membrane trafficking to attenuate tumorigenesis in glioblastoma subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5433. doi:10.1158/1538-7445.AM2017-5433

Background: Entrectinib is a potent, CNS-penetrant, oral inhibitor of the tyrosine kinases TRKA/B/C (encoded by the genes NTRK1/2/3, respectively), ROS1, and ALK with IC50s < 2 nM (biochemical kinase assay). Two Phase 1 studies ALKA-372-001 and STARTRK-1 have enrolled more than 130 patients with advanced or metastatic solid tumors harboring TRKA/B/C, ROS1, or ALK molecular alterations, with or without CNS disease. Previously, we reported an objective response rate of 79% in 24 tyrosine kinase inhibitor-naïve patients with TRK, ROS1, or ALK gene fusions who were treated at doses that achieved therapeutic exposures consistent with the Recommended Phase 2 Dose (RP2D) (Drilon et al, AACR 2016). Entrectinib was well tolerated, with predominantly Grades 1 or 2 adverse events that were reversible with dose modification. Responses were observed in NSCLC, colorectal cancer, mammary analog secretory carcinoma, melanoma, and renal cell carcinoma, as early as 4 weeks after starting treatment and lasting as long as > 2 years. Notably, a complete CNS response was achieved in a patient with SQSTM1-NTRK1-rearranged lung cancer. Methods: STARTRK (Studies of Tumor Alterations Responsive to Targeting Receptor Kinases)-2 is a potentially registration-enabling Phase 2 basket study of entrectinib for the treatment of patients with advanced solid tumors that harbor a TRK, ROS1, or ALK gene fusion. In order to determine enrollment eligibility and assignment to a specific tumor type basket, patients are screened for gene fusions either locally, including by ctDNA, or centrally at Ignyta’s CLIA/CAP diagnostic laboratory using next generation sequencing. The study’s eligibility criteria were designed to maximize enrollment of these rare patients by allowing CNS disease, Eastern Cooperative Oncology Group (ECOG) performance status 2, and any prior line of therapy, with the exception of TRK, ROS1, or ALK inhibitors. Patients with ALK- or ROS1-rearranged NSCLC who had previously been treated with crizotinib and experienced CNS-only progression are also eligible. In addition, a “non-evaluable” basket allows enrollment of patients confirmed to have gene fusions who do not meet all the inclusion or exclusion criteria. Entrectinib is administered orally on a continuous daily dosing regimen, at a dose of 600 mg once-daily in repeated 4-week cycles. Safety is assessed by monitoring of adverse events, laboratory tests, and clinic visits. Tumor assessments (computed tomography (CT) or magnetic resonance imaging (MRI)) of the chest, abdomen, pelvis (depending on tumor type), plus bone and/or brain as applicable, are performed at the end of Cycle 1 and every 8 weeks thereafter. All CT and MRI scans are read by a central independent imaging laboratory using the Response Evaluation Criteria In Solid Tumors (RECIST) v1.1 and the Response Assessment in Neuro-Oncology Criteria (RANO) or RANO - Brain Metastases (RANO-BM), as applicable, for patients with primary or secondary CNS disease, respectively. Blood and tissue are collected at the time of progression for biomarker analyses for potential mechanisms of resistance to entrectinib. Patients remain on study treatment until documented radiographic progression as assessed by blinded independent central review (BICR), development of unacceptable toxicity, or withdrawal of consent. Citation Format: Alexander Drilon, Kamalesh Kumar Sankhala, Stephen V. Liu, Byoung Chul Cho, Collin Blakely, Cheng E. Chee, Marwan Fakih, Jonathan Polikoff, Zachary Hornby, Lisa Schechet, David Luo, Edna Chow Maneval, Pratik S. Multani, Robert C. Doebele. STARTRK-2: A global phase 2, open-label, basket study of entrectinib in patients with locally advanced or metastatic solid tumors harboring TRK, ROS1, or ALK gene fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT060. doi:10.1158/1538-7445.AM2017-CT060

Medulloblastoma (MB) is the most common malignant brain tumor in children and accounts for 20% of pediatric CNS tumors. Following surgery and treatment with chemotherapy and radiation, MBs recur in a significant proportion of patients. Moreover, survivors experience comorbidities related to the tumor and/or its treatment. New insights into MB tumor pathogenesis are needed for improving the survivorship and outcome of MB. Epigenetic silencing, including histone modification and DNA methylation, is an important tumorigenic mechanism in MB. However, its role in cancer immunopathology is poorly understood. Using datamining and immunohistochemistry analysis, we show that B7-H3 (CD276), an important immune checkpoint member of the B7 and CD28 families, is highly upregulated in all four molecular subtypes of MB compared to normal tissue. Based on the clinical success of inhibitory immune checkpoint blockades (CTLA-4, PD-1, and PD-L1), CD276 appears to be a promising therapeutic target for cancer immunotherapy. Moreover, we observed that increased expression of CD276 correlates significantly with the expression of Enhancer of Zeste Homologue 2 (EZH2), a histone H3-methylating (H3K27me3) catalytic subunit in the human MB specimens. We hypothesize that combined targeting of CD276 and EZH2 may have greater impact on tumor progression and immune cell response in MB. Simultaneously, we performed LC/MS analysis to identify novel interaction partners of CD276 in MB tumor. These findings taken together suggest that CD276 is strongly associated with several epigenetic markers, indicating a strong relationship between cancer immune evasion and epigenetic mechanisms. Citation Format: Maheedhara R. Guda, Jose R. Castellanos, Collin M. Labak, Ramadevi Sanam, David J. Daniels, Sarah E. Martin, Julian J. Lin, Andrew J. Tsung, Kiran K. Velpula, Swapna Asuthkar. Epigenetic targeting of CD276 expression in medullolbastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2004. doi:10.1158/1538-7445.AM2017-2004

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-β signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer. (Cancer Res 2006; 66(12): 6421-31)

The vast majority of patients with lung adenocarcinomas harboring activating mutation in EGFR respond to EGFR tyrosine inhibitors (TKI) (i.e. erlotinib). However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. We recently identified activation of the NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung adenocarcinoma. However, the role of NF-κB activation in mediating EGFR TKI acquired resistance in lung adenocarcinoma is not well defined. We have found that erlotinib treatment of EGFR TKI sensitive lung adenocarcinoma cell lines promotes the rapid phosphorylation and degradation of IκB (inhibitor of NF-κB) and subsequent activation of the NF-κB subunit RelA. RelA activation is accompanied by transcriptional activation of downstream NF-κB target genes, including IL6. Prolonged exposure of EGFR TKI sensitive lung adenocarcinoma cell lines to erlotinib leads to EGFR TKI acquired resistance that is accompanied by sustained NF-κB activation and IL6 expression. EGFR TKI acquired resistance can be overcome by treating cells with erlotinib in combination with PBS-1086 (rel∼MD, Inc.), a direct Rel inhibitor. Furthermore, concomitant treatment of EGFR TKI sensitive lung adenocarcinoma cells with erlotinib + PBS-1086 prevents the development of EGFR TKI acquired resistance. Together, these results demonstrate the molecular basis for the synthetic lethality of combined EGFR and NF-κB inhibition and provide mechanism-based rationale for polytherapies against both EGFR and NF-κB to enhance response in lung adenocarcinoma patients. Broadly, our findings provide novel insights into the biological and clinical consequences of the context-specific and dynamic functional interplay between EGFR and NF-κB signaling. Citation Format: Collin M. Blakely, Evangelos Pazarentzos, Saurabh Asthana, Victor Olivas, Irena Tan, Timothy Fouts, Jeffrey Meshulam, Trever G. Bivona. Erlotinib induces NF-kappa B dependence that promotes EGFR tyrosine kinase inhibitor resistance in lung adenocarcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1836. doi:10.1158/1538-7445.AM2014-1836

Treatment of patients with lung cancers that harbor activating mutations in the kinase domain of EGFR (e.g. L858R, in frame Exon19 deletion), which account for 15-20% of lung adenocarcinomas, with the EGFR tyrosine kinase inhibitor (TKI) erlotinib, causes tumor regression in many patients. However, the magnitude of tumor regression is variable and responses are short-lived with a median duration of 9-12 months. Thus inhibition of mutant EGFR activity by treatment with erlotinib results in incomplete responses and is not curative in the majority of EGFR-mutant lung cancer patients. While mechanisms of acquired resistance to EGFR TKI treatment are well defined, mechanisms of de novo resistance, or incomplete initial response to EGFR TKI therapy, remain uncharacterized. We recently identified activation of the NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) signaling pathway as a key mediator of de novo resistance to EGFR TKI therapy in cell lines and tumor xenograft models of lung cancer. Furthermore, patients with EGFR-mutant lung cancers that demonstrate elevated IkB (NF-kB inhibitor) expression, and consequently low NF-kB activity, exhibit improved responses to EGFR TKIs compared to patients with low IkB levels. NF-kB is a family of transcription factors that regulates numerous biological processes, including cell proliferation and apoptosis, as well as a critical mediator of both innate and adaptive immune cell activity. We hypothesized that pharmacologic inhibition of NF-kB activity would enhance the efficacy of EGFR TKI in EGFR-mutant lung cancer. Using a novel rel (reticuloendotheliosis) inhibitor of NF-kB, PBS-1086 (rel∼MD, Inc.), we found that treatment of human lung cancer cells and tumor xenografts that harbor an EGFRL858R activating mutation (11-18), yet are EGFR TKI resistant, with erlotinib in combination with PBS-1086 induces tumor cell apoptosis and tumor regression. Erlotinib + PBS-1086 treatment results in decreased binding of the NF-kB subunit RelA to the IL6 promoter and a significant decrease in IL6 mRNA expression. IL6 has previously been shown to promote EGFR-mutant lung adenocarcinoma cell proliferation and survival, and its downregulation by PBS-1086 represents a possible mechanism of action of the compound. Using CC10-rtTA; Tet-Op-EGFR-mutant transgenic mouse models of lung adenocarcinoma, we have shown that treatment of mice with PBS-1086 enhances erlotinib-induced tumor regression. We believe that these results will guide future clinical trials of NF-kB inhibitors in combination with erlotinib to improve outcomes in selected EGFR-mutant lung cancer patients. Citation Format: Collin M. Blakely, Victor Olivas, Jie Zhang, Trever G. Bivona. Pharmacologic inhibition of NF-kappaB overcomes de novo resistance to erlotinib in models of EGFR-mutant lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4631. doi:10.1158/1538-7445.AM2013-4631

We, and others, have previously reported that macrophages infiltrating mammary adenocarcinomas foster late-stage cancer progression1-3, and that targeted therapies diminishing macrophage presence in tumors, and improve response to standard chemotherapy (CTX) resulting in slowed primary tumor growth, reduced pulmonary metastasis and increased overall survival (OS) of tumor-bearing mice4,5. These experimental studies correlate with clinical findings indicating that high densities of CD68+ myeloid cells correlate with reduced OS in patients with breast cancer6. Malignant mesotheliomas (MM), a rare cancer that can develop as a result of prior asbestos exposure, is highly resistant to conventional cytotoxic therapy7, and in the pleural space is associated with significant densities of macrophages that also correlate with decreased OS8. To investigate a functional role for macrophages in fostering neoplastic progression of MM, and/or in limiting response to CTX, we investigated the efficacy of several antagonists targeting the colony stimulating factor-1 (CSF1)/CSF-1 receptor (CSF1-R) CSF-1R signaling cascade to reveal the functional significance of macrophages in MM, and to determine if this class of agents exhibited efficacy as monotherapy, or in combination with CTX, to improve OS of tumor-bearing mice. Using a syngeneic orthotopic mouse model for MM, our results indicate that reducing the presence of macrophages in MM significantly slows early, as well as late-stage MM growth, and improves response to standard-of-care CTX. These experimental findings indicate a protumoral role for macrophages in MM, and support clinical investigation of targeted therapies, in combination with CTX, for treatment of patients with MM. 1. Lin et al., J Exp Med 193, 727-740 (2001). 2. DeNardo et al., Cancer Cell 16, 91-102 (2009). 3. Gocheva et al., Genes Dev 24, 241-255 (2010). 4. DeNardo et al., Cancer Discov 1, 54-67 (2011). 5. Shree et al., Genes and Development in press(2011). 6. Campbell et al., Breast Cancer Res Treat 128, 703-711 (2011). 7. Sugarbaker et al., Expert Rev Respir Med 4, 363-372 (2010). 8. Burt et al., Cancer 117, 5234-5244 (2011). The authors acknowledge generous support from the NIH/NCI, Dept of Defense (DoD) Era of Hope Scholar Award Expansion Award, Susan G. Komen Fndt, Breast Cancer Research Fndt, and an Investigator-Initiated Research Award in Mesothelioma from the DoD. Citation Format: Collin Blakely, Tina Bose, Melissa Wheeler, Raphael Bueno, David Sugarbaker, David Jablons, V. Courtney Broaddus, Lisa Coussens. Macrophages foster neoplastic progression of malignant mesothelioma and limit response to chemotherapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4723. doi:10.1158/1538-7445.AM2013-4723

Macrophages within the tumor microenvironment (TME) may contribute to tumor chemoresistance. We have found that macrophages constitute a significant percentage of the infiltrating leukocytes in mesothelioma, a highly chemoresistant tumor. We asked whether reprogramming of macrophage phenotype from a Th2 (pro-tumor) to a Th1 (anti-tumor) phenotype would alter the mesothelioma chemoresponsiveness. To address this, we used 3D organotypic models of mesothelioma growth: 1) multicellular spheroids, with mesothelioma cells grown alone or co-cultured with macrophages derived from peripheral blood monocytes, and 2) primary tumor fragment spheroids derived from small fragments of freshly isolated human mesothelioma. Macrophages were incubated with Th1 (LPS & IFNγ) or Th2 (IL-4 &IL-13) cytokines prior to co-culture with mesothelioma spheroids, followed by exposure to standard-of-care chemotherapy, cisplatin plus pemetrexed. Gene expression was evaluated in macrophages to affirm Th1-type (TNF, IL-12, IFNα) versus Th2-type (CD206, IL10) programming. In both spheroid approaches, Th1-type macrophage programming significantly increased the apoptotic response of mesothelioma cells to chemotherapy. Moreover, when CSF1R signaling in macrophages was inhibited by incubation with a small molecular weight kinase antagonist, GW2850, chemoresponsiveness was significantly increased as evidenced by increased presence of apoptotic tumor cells in spheroids. Enhanced chemoresponsiveness of mesothelioma tumor cells was dependent on presence of macrophages because incubation of tumor cells with Th1-type cytokines or GW2850 in the absence of macrophages was without effect. We conclude that manipulating the TME may be a promising therapeutic approach in mesothelioma. This work was supported by a DOD Mesothelioma Program grant PR80717 to Broaddus and Coussens and a T32 Training Grant to Battula and Blakely. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-251. doi:1538-7445.AM2012-LB-251

Malignant pleural mesothelioma (MPM) is a debilitating, incurable cancer that exhibits a high degree of resistance to standard chemotherapy; thus, we sought to identify novel therapeutic targets for treatment. In the vast majority of cases, MPM is associated with exposure to asbestos fibers, which result in a chronic pro-inflammatory state. As such, we hypothesized that MPMs were infiltrated by leukocytes possessing tumor-potentiating activities. To address this, we evaluated MPMs, resected from 26 patients, by flow cytometry and found that tumors were highly infiltrated with macrophages (31 ± 4.6% of total CD45+), a percentage that is significantly higher that that observed in other thoracic malignancies (NSCLC cancer, 9%; esophageal, 4%). Since recent experimental data has revealed that tumor-associated macrophages (TAMs) secrete proangiogenic, prosurvival, and proinvasive factors that foster tumor progression, we evaluated macrophage-depletion in MPM as a novel therapeutic strategy. Using a syngeneic murine transplantation model and liposomal-encapsulated clodronate that efficiently deplete phagocytic macrophages, as monotherapy, and in combination with chemotherapy, we revealed a significant decrease in tumor growth and a decrease in tumor-burden in tumor-bearing mice depleted of macrophages. Furthermore, when macrophage-depletion was combined with the cytotoxic agents gemcitabine and TRAIL, an even greater reduction in tumor burden was observed as compared to mice treated with either agent alone. These studies indicate that: 1) mesothelioma-associated macrophages provide a protumor function, and 2) depletion of mesothelioma-associated macrophages may improve efficacy of cytotoxic chemotherapy. Ongoing studies to reveal the effect of macrophage-depletion plus chemotherapy in limiting MPM development in NF2ko/+ mice exposed to asbestos are underway. This work was supported by an NIH T32 training grant to Dr. Blakely, and by a DoD Mesothelioma Program grant (PR080717) to Drs. Broaddus and Coussens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 454. doi:10.1158/1538-7445.AM2011-454