bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 928: Preventing melanoma from healing itself after therapy-induced immune attack through IPRES blockade
Authors: Piva Marco , Sun Lu , Moriceau Gatien , Yang Zhentao , Ribas Antoni , Hugo Willy , Lo Roger S.
Published: August 2019

Over the past few years, therapeutics development for melanoma has led the oncology field, including the development of mutant BRAF-targeted therapies (e.g., BRAF inhibitor (BRAFi)) and immune checkpoint blockade therapies (e.g., anti-PD-1 or aPD-1, aPD-L1, etc). Based on the complementary qualities of aPD-1/L1 immunotherapy (long duration of response) and MAPKi (high rate of initial response), there are currently clinical trials testing the combination of the two. However, despite a more durable tumor suppression, resistance still eventually develops in patients treated by the combination (ref: KEYNOTE-022). My earlier study reported significant intra-patient and intra-tumoral heterogeneity which affect patient-specific responses to therapy. Furthermore, I also observed complex transcriptomic and tumor microenvironment reprogramming associated with resistance to targeted- or immuno-therapies in melanoma. However, the increased immune/stromal cells in the tumor under therapy has prevented us from definitively accounting the source of such reprogramming. To address this need, I utilized the novel single cell transcriptomic analysis to discover therapy-response-associated gene programs across different types of cells within a tumor. In murine mutant BRAF melanoma YUMM1.7, regressing/responding tumors on the 6th day of BRAFi treatment showed significant infiltration of the T cells and other immune cell population and, by the 15th day, the tumor showed activation of IPRES related processes. Combination of BRAFi and aPD-L1 in YUMM1.7 increased the proportion of lymphoid population in the tumor on the 7th day but only incrementally delayed the development of BRAFi resistance. Inhibition of IPRES-related processes (anti-VEGF and/or anti-TGFB) further delayed BRAFi resistance but no complete responses were observed. However, when we employed a UV-mutated subline of the same model, named the YUMMER (Bosenberg lab, Yale), combination of BRAFi, aPD-L1 and IPRES inhibition started to result in durable complete responses in our mouse model. We are now in midst of analyzing the differential response that we observed with this model; our current working hypothesis is that YUMMER induces higher level of antigen-specific antitumor response, which is augmented by the addition of immune checkpoint blockade and IPRES suppression. The results from our study will eventually pave the way to a novel therapeutic combination to overcome melanoma.

Citation Format: Marco Piva, Lu Sun, Gatien Moriceau, Zhentao Yang, Antoni Ribas, Willy Hugo, Roger S. Lo. Preventing melanoma from healing itself after therapy-induced immune attack through IPRES blockade [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 928.
Abstract 931: The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer
Authors: Kadi Najwa El , Wang Luo , Davis April , Cooke Alexander , Vadnala Varun , Korkaya Hasan , Kalemkerian Gregory P. , Hassan Khaled A.
Published: August 2019

Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based.

Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFκB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFκB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFκB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression.

Conclusion: In EGFR driven lung adenocarcinoma, NFκB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance.

Citation Format: Najwa El Kadi, Luo Wang, April Davis, Alexander Cooke, Varun Vadnala, Hasan Korkaya, Gregory P. Kalemkerian, Khaled A. Hassan. The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer [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 931.
Abstract 955: Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant NSCLC
Authors: Kudinov Alexander , Makhov Petr , Faezov Bulat , Bychkov Igor , Deneka Alexander , Nicolas Emmanuelle , Caid Kathy Q Q. , Brebione Rohan , Avrilf Eleanor , Nikonova Anna S. , Serebriiskii Ilya G. , Borghaei Hossein , Golemis Erica A. , Boumber Yanis
Published: August 2019

Musashi-2 (MSI2) is an RNA-binding protein that regulates mRNA translation. We recently established that MSI2 is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression and drives NSCLC metastasis, in part based on activity supporting a TGF-beta/SMAD3/claudin signaling cascade. Here, reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+;P53R172HΔG/+ murine NSCLC cell lines identified a significant upregulation of HER3 (ERBB3) upon MSI2 depletion. Negative MSI2-dependent regulation of ERBB3 protein was confirmed in multiple murine and human NSCLC models, based on analysis of MSI2 depletion or overexpression. Further, MSI2 positively regulated expression of the epidermal growth factor receptor (EGFR) protein in the same models. Comparing EGFR and KRAS driven models, we found that MSI2 depletion significantly impairs cell proliferation and clonogenicity only in EGFR-mutant NSCLC cell lines. Using RNA immunoprecipitation analysis coupled with qPCR, we show that MSI2 directly binds to EGFR and, to a lesser extent, to HER3 mRNA. MSI2 mRNA binding was correlating with the presence of predicted MSI2 binding sites in corresponding mRNAs. Taken together, this data suggest direct regulation of EGFR protein expression by MSI2 at post transcriptional level. NSCLC lung tissue microarray immunohistochemistry analysis revealed that MSI2 total positivity by H-score, 2+/3+ and 3+ positivity correlated with EGFR 3+ staining. Finally, EGFR inhibitors erlotinib and afatinib synergized with MSI2 depletion in EGFR mutant models, suggesting that therapeutic targeting of MSI2 could be of clinical value, especially in EGFR-mutant lung cancer. In vivo experiments of erlotinib treatments of wild type or MSI2 depleted tumors are ongoing and these results will be presented.

Citation Format: Alexander Kudinov, Petr Makhov, Bulat Faezov, Igor Bychkov, Alexander Deneka, Emmanuelle Nicolas, Kathy Q Q. Caid, Rohan Brebione, Eleanor Avrilf, Anna S. Nikonova, Ilya G. Serebriiskii, Hossein Borghaei, Erica A. Golemis, Yanis Boumber. Musashi-2 regulates EGFR expression in NSCLC, cell proliferation and survival, response to EGFR inhibitors in EGFR-mutant 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 955.
Abstract 974: The molecular and genomic drivers of immunologically cold tumors: Novel discoveries from pan-cancer analysis
Authors: Han Guangchun , Wang Ruiping , Shen Jianfeng , Singh Pankaj , Zhang Shaojun , Rao Arvind , Gao Jianjun , Lazar Alexander , Futreal Andrew , Peng Guang , Rai Kunal , Wang Linghua
Published: August 2019

Immune-checkpoint blockade (ICB) is able to elicit remarkable immune response but currently the durable response is limited to a minority of cancer patients. Recent evidence suggests that a prerequisite for the efficacy of ICB, for example the PD-1 inhibitor, is T-cells infiltration in the tumor microenvironment (TME). The immunologically ‘quite’ and T-cell depleted (immune ‘desert’) tumors are less likely respond to ICB. However, we still lack a detailed understanding of the genomic and molecular determinants that drive the ‘cold’ immune phenotypes. We here performed a comprehensive pan-cancer analysis to define and characterize the immunologically ‘hot’ (inflamed) and ‘cold’ (quite or T-cell depleted) tumors by high-dimensional, multi-platform data integration of 9,887 tumors from The Cancer Genome Atlas (TCGA). This study provided an unprecedented catalog of the molecular and genomic drivers of immune ‘cold’ tumors by analyzing the immune cell composition, patterns of their spatial organization within the TME, the dysregulated chemokines networks, somatic mutations, DNA copy-number alterations, aneuploidy status, differentially expressed immune regulatory genes, and enriched signaling pathways. We showed that the immunologic status is highly heterogeneous within and across TCGA tumor types and subtypes. We integrated the information of T-cell spatial organization with our immune phenotypes and found that the ‘band-like’ spatial organization (T cells clustered at the invasive margin of the tumor and didn’t get into the tumor body) pattern is strongly associated with the ‘cold’ phenotype, particularly in colon and stomach cancers. Our analysis revealed a number of other factors that are associated with the ‘cold’ phenotype by either preventing T-cell infiltration or promoting T-cell dysfunction, and we performed functional validation on several targets of interest. This study provided an in-depth understanding of the genomic and molecular determinants of immunologically ‘cold’ tumors and suggested potential biomarkers that can be targeted for the development of novel combination therapy strategies to convert the ‘cold’ tumors into ‘hot’ ones and improve the odds of cancer immunotherapy success.

Citation Format: Guangchun Han, Ruiping Wang, Jianfeng Shen, Pankaj Singh, Shaojun Zhang, Arvind Rao, Jianjun Gao, Alexander Lazar, Andrew Futreal, Guang Peng, Kunal Rai, Linghua Wang. The molecular and genomic drivers of immunologically cold tumors: Novel discoveries from pan-cancer analysis [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 974.
Abstract 981: Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design
Authors: Andersen Jannik N. , McRiner Andrew J. , Fouser Lynette A. , Zhang Junyi , Arora Shilpi , Cordeau Michael , Zhang Ying , Cuozzo John , Briskin Michael , Clark Matt , Ezzeddine Diala
Published: August 2019

Since the first small molecule proteolysis targeting chimera (PROTAC) was reported about a decade ago, great progress has been made in the field of targeted protein degradation. Specially designed, small molecules can recruit the ubiquitin-proteasome system (UPS) to tag a protein of interest (POI) for degradation. Based on the ability to knock down a therapeutic POI (instead of inhibiting the target protein activity), this new modality has emerged as a paradigm-shifting approach and opened new avenues for small molecule drug discovery. At Xios Therapeutics, we have applied targeted protein degradation to a number of immuno-oncology (IO) drug targets and we present here the strategy and lessons learned from building our PROTAC platform in collaboration with X-Chem. Specifically, we have leveraged a vertical integration of DNA-encoded library screening (DEL), structural biology, medicinal chemistry, biochemical binding assays and cellular biomarker readouts for the rapid identification of cell potent degraders. We exemplify a modular, ‘fit-for-purpose’ PROTAC matrix that allows for rapid exploration of optimal E3 ligase-binders conjugated to a POI-binder using either existing or novel ligands identified via DEL. We delineate the structure-activity/property relationship (SAR and SPR) analysis of linker with VHL- and CRBN-based binders for a promising IO target achieving potent protein degradation (>90% degradation and nM DC50 potency) and pathway inhibition in cancer cells. Notably, our affinity-based screening of chemical libraries of unprecedented size (~200 billion molecules) with a priori knowledge of the vector point of attachment from the DNA barcode directly informs the rational design of bifunctional PROTAC molecules. In conclusion, our integrated approach allows us to find new, unexplored compound binding sites for both E3 ligases- and POI-binders that can be utilized by the PROTAC platform to create potent selective degraders and to access targets that have previously been considered undruggable.

Citation Format: Jannik N. Andersen, Andrew J. McRiner, Lynette A. Fouser, Junyi Zhang, Shilpi Arora, Michael Cordeau, Ying Zhang, John Cuozzo, Michael Briskin, Matt Clark, Diala Ezzeddine. Degradation of immuno-oncology targets via proprietary PROTAC platform integrating DNA-encoded library technology and rational drug design [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 981.
Abstract 986: High content analysis of 2D and 3D oncology models for target and phenotypic drug discovery
Authors: Lachize Servane , Bospoort Rhea van de , Silva Marta da Silva da , Steentraten Niki van der , Gowers Ian , Clark Roger , DeGroot Jeroen , Waddell Ian , Schiewe Hajo , Jain Shushant
Published: August 2019

Charles River (CR) has developed many disease-relevant cellular models to screen small molecules and genetic modifiers for target and phenotypic based drug discovery. Our expertise and portfolio in bespoke oncology model development (e.g. patient derived xenografts) covers a wide variety of target classes such as inhibitors of cell cycle, cellular motility and migration and transcription factor translocation. Complex cellular models derived from primary cells, patient derived embryonic stem cells and induced pluripotent stem cells have been adapted for phenotypic screening. Models have been further developed to produce physiologically relevant multicellular structure co-cultures. Combining complex biological systems with multiparametric high content analysis provides detailed single cell quantification of cellular and subcellular biology. Such validated High Content assays can be applied to both target and phenotypic based drug-discovery platforms to support preclinical drug discovery and improve translation of targets and compounds to the clinic.

Citation Format: Servane Lachize, Rhea van de Bospoort, Marta da Silva da Silva, Niki van der Steentraten, Ian Gowers, Roger Clark, Jeroen DeGroot, Ian Waddell, Hajo Schiewe, Shushant Jain. High content analysis of 2D and 3D oncology models for target and phenotypic drug discovery [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 986.
Abstract CT001: Biomarkers of systemic inflammation associated to reduced clinical activity of atezolizumab monotherapy in patients with metastatic triple negative breast cancer
Authors: Li Yijin , Fassò Marcella , Emens Leisha A. , Molinero Luciana
Published: August 2019

Background: Immune checkpoint blockade by anti-PD-L1 antibody atezolizumab has demonstrated clinical benefits in metastatic triple negative breast cancer (mTNBC). IL-6 and IL-8 myeloid inflammation is linked to poor prognosis in cancer patients treated with chemotherapy, but its association with single agent atezolizumab-treated patients with mTNBC remains unknown. In this study, we investigated the association of biomarkers (BM) of systemic inflammation with clinical outcomes in patients with mTNBC treated with atezolizumab monotherapy.

Methods: Baseline pre-treatment plasma samples were collected from mTNBC patients enrolled in the Phase I atezolizumab monotherapy clinical trial PCD4989g (NCT01375842, n = 116). IL-6, IL-8 and CRP were tested by ELISA or Luminex assays and their levels were assessed for association with baseline clinical demographic characteristics and atezolizumab clinical activity for response rate (ORR), progression free (PFS) and overall survival (OS).

Results: Baseline IL-6, IL-8, and CRP levels were positively associated with the clinical prognostic traits ECOG performance status (>0), presence of liver metastases, large size of target lesions (>=6.5cm), and increased LDH (>=1.5xULN). Elevated baseline IL-6 and CRP, but not IL-8, were also linked with later lines of therapy (>=2). Univariate analyses showed that IL-6 (>=15pg/ml), IL-8 (>=11.4pg/ml) and CRP (>=3mg/L) were associated with reduced OS and PFS (Table), but only CRP was associated to reduced ORR. Multivariate analyses considering the aforementioned clinical demographic variables showed that IL-6 (HR 2.00 [1.16-3.38]); and CRP (HR 2.74 [1.49-5.28]), but not IL-8 (HR 1.07 [0.78-1.74]), were associated with OS.

Conclusion: The systemic IL-6/CRP inflammatory axis is an independent factor linked with poor outcomes of mTNBC patients treated with atezolizumab monotherapy and may play unique roles in affecting the anti-tumor activity in this hard to treat patient population.

Table (univariate)BMLow mPFSBMHigh mPFSPFS HR (CI 95%)BMLow mOSBMHigh mOSOS HR (CI 95%)IL-6 (>=15pg/ml)1.45 mo1.35 mo1.87 (1.23-2.83)12.6 mo2.6 mo2.55 (1.63-4.01)IL-8 (>=11.4pg/ml)1.94mo1.34 mo2.00 (1.36-2.95)12.6 mo6.42 mo2.05 (1.34-3.14)CRP (>=3mg/L)2.50 mo1.35 mo2.07 (1.31-3.25)20.5 mo6.9 mo3.33 (1.91-5.79)

Citation Format: Yijin Li, Marcella Fassò, Leisha A. Emens, Luciana Molinero. Biomarkers of systemic inflammation associated to reduced clinical activity of atezolizumab monotherapy in patients with metastatic triple negative breast cancer [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 CT001.
Abstract CT028: Drug-drug interaction potential of EZH2 inhibitor tazemetostat (TAZ)
Authors: Amengual Jennifer E. , Schwartz Gary K. , Inclan Lora , Kumar Abhijeet , Robles-Medina Patricia , Singh-Kandah Shahnaz , Suttle Benjamin , Beelen Melissa , Persky Daniel
Published: August 2019

TAZ, a selective, oral EZH2 inhibitor, is in the phase 2 clinical investigation stage for the treatment of advanced solid tumors (AdvST) and B-cell non-Hodgkin lymphoma (NHL). Drug-drug interaction (DDI) potential of TAZ was investigated in study EZH-105, a phase 1, open-label, 2-part study in adults with AdvST or NHL. In Part A, patients received TAZ 400 mg twice-daily (BID) days 1-24, then 800 mg BID continuously. Moderate CYP3A inhibitor fluconazole 400 mg was administered once daily (QD) days 16-19. Serial blood samples for pharmacokinetic analysis were collected on days 15 and 19. Patients in Part B received TAZ 800 mg BID continuously starting day 2. The CYP2C8 probe repaglinide 0.25 mg and CYP2C19 probe omeprazole 20 mg were administered on day 1 and 1 hour following TAZ administration on day 16. Omeprazole 20 mg QD continued days 17-19. Serial blood samples were collected on days 1, 16, and 19. DDIs were evaluated based on AUC and Cmax. Other primary endpoints included safety of TAZ. As of August 14, 2018, 27 patients were enrolled (n=17 AdvST, n=10 NHL). Median age was 60 years. Preliminary pharmacokinetic results are displayed in the Table. Median treatment exposure was 70 days in Part A and 57 days in Part B. Two patients, both in Part B, discontinued due to treatment emergent adverse events (TEAEs). Grade ≥3 TEAEs were reported in 12 patients. The most frequent treatment-related TEAEs (any grade) included diarrhea (n=6), fatigue (n=6), and nausea (n=3). In conclusion, the likelihood of a clinically significant DDI in the presence of TAZ is minimal.

Table.Preliminary study EZH-105 PK resultsPart A. The effect of fluconazole on TAZParameterTAZ alone Day 15 Mean (%CV)TAZ + fluconazole Day 19 Mean (%CV)TAZ, n=14Cmax, ng/ml426 (95)968 (80)AUC0-12h, ng*h/mL1590 (81)5180 (66)Part B. The effect of TAZ on REP and OMPREP + OMP Day 1 Mean (%CV)REP + OMP + TAZ Day 16 Mean (%CV)REP, n=12Cmax, ng/ml5 (109)10 (78)AUC0-7h, ng*h/mL9 (110)17 (65)OMP, n=12Cmax, ng/ml263 (266)220 (250)AUC0-7h, ng*h/mL677 (340)558 (231)Part B. The effect of OMP on TAZTAZ prior to OMP Day 16 Mean (%CV)TAZ + OMP Day 19 Mean (%CV)TAZ, n=9Cmax, ng/ml613 (91)663 (68)AUC0-12h, ng*h/mL2200 (58)2820 (62)AUC, area under the concentration curve; CI, confidence interval; Cmax, maximum plasma concentration; CV, coefficient of variation; REP, repaglinide; OMP, omeprazole; PK, pharmacokinetic; TAZ, tazemetostat.

Citation Format: Jennifer E. Amengual, Gary K. Schwartz, Lora Inclan, Abhijeet Kumar, Patricia Robles-Medina, Shahnaz Singh-Kandah, Benjamin Suttle, Melissa Beelen, Daniel Persky. Drug-drug interaction potential of EZH2 inhibitor tazemetostat (TAZ) [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 CT028.
Abstract CT047: Phase I trial of intravesical Bacillus Calmette-Guérin combined with intravenous Pembrolizumab in high grade nonmuscle invasive bladder cancer
Authors: Alanee Shaheen , Zawahry Ahmed El , Sana Sherjeel , McVary Kevin , Robinson Kathy , Rao Krishna A.
Published: August 2019

PURPOSE: A phase I trial of intravesical bacillus Calmette-Guérin (BCG) in combination with systemic pembrolizumab was conducted in patients with high grade non-muscle invasive bladder cancer who had persistent or recurrent disease after failing treatment with at least 2 courses of intravesical therapy (one of which had to contain BCG) or BCG followed by maintenance BCG. The primary objective was to determine the safety of this combination. Secondary end points included response to treatment at 19 weeks (end of treatment) and 3 months post treatment.

MATERIALS AND METHODS: A total of 9 patients with recurrent/persistent high-grade non-muscle invasive bladder cancer after at least two courses of intravesical therapy or one course of BCG treatment followed by one course of maintenance BCG were enrolled in the study. Six doses of pembrolizumab were given every 3 weeks over 16 weeks given concurrently with 6 weekly doses of BCG beginning at week 7. Patient safety was evaluated during and for 30 days following pembrolizumab treatment. Preliminary combination efficacy was determined at 19 weeks using cystoscopy. Bladder biopsy was performed in patients with suspicious lesions.

RESULTS: The combination of BCG and pembrolizumab was well tolerated at both 100mg and 200mg fixed doses. Fatigue and cystitis-type symptoms (burning, spasm, urgency, sensitivity, and frequency) were the most common adverse events and all cases were either grade 1 or 2. Two patients died during the trial period. One patient died due to progression of low grade upper urinary tract transitional cell carcinoma. The other patient died after cystectomy (for progressive disease) due to causes unrelated to the trial treatment. Of the 9 patients treated, 6 (78%) had no evidence of disease in the bladder and 1 had a mixed response (progression in the upper urinary tract only) at 19 weeks (end of treatment) while 5 (56%) remained disease-free at the 3 month follow up.

CONCLUSIONS: The combination of BCG and pembrolizumab was well tolerated with subjects exhibiting the expected adverse effects associated with the use of both medications. Intravesical BCG and pembrolizumab may have clinical activity in non-muscle invasive bladder cancer recurring after repeated intravesical therapy. .A phase III trial is now open and enrolling in the United States and other countries (NCT03711032).

Citation Format: Shaheen Alanee, Ahmed El Zawahry, Sherjeel Sana, Kevin McVary, Kathy Robinson, Krishna A. Rao. Phase I trial of intravesical Bacillus Calmette-Guérin combined with intravenous Pembrolizumab in high grade nonmuscle invasive bladder cancer [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 CT047.
Abstract CT064: Intranasal brain delivery using NEO100 for recurrent glioblastoma
Authors: Chen Thomas C. , Wagle Naveed , Peereboom David , Taylor Lynne
Published: August 2019

NO CHANGES TO ORIGINAL ABSTRACT

NEO100 is a GMP quality, purified perillyl alcohol (POH) which has been undergoing clinical trials under IND approval (144,595) entitled "Open label, Phase I/IIa dose escalation study of safety and efficacy of NEO100 in recurrent grade IV glioma." It was granted orphan drug and fast track status by the FDA. Inclusion criteria include temozolomide failure, bevacizumab naïve, tumor less than 3x3 cm, and Karnofsky score greater than or equal to 60. Patients were enrolled from University of Southern California (USC), Cleveland Clinic, and University of Washington. Total number of patients to be enrolled for Phase I is 12 with four dose escalating cohorts of 3 patients per cohort. The remaining 31 patients would be enrolled after maximal tolerated dosage (MTD) for Phase IIa. The delivery is intranasal via a nasal nebulizer using the concept of nasal brain delivery via the olfactory and trigeminal nerves for delivery to the brain and tumor. To date, 8 patients have been enrolled. Cohort 1 received 96 mg/dose qid, Cohort 2 received 144 mg/dose qid, cohort 3 received 192 mg/dose qid, and cohort 4 will receive 288mg/dose qid. No toxicities were detected. Inhalation was very well tolerated. The duration of the trial is for 6 months. To date, 3 patients have not shown evidence of progression for greater than 6 months (one patient 1 year (cohort 2), one patient 7 months (cohort 3), one patient 6 months) (cohort 3). This is the first FDA approved trial for intranasal delivery to malignant gliomas. Although preliminary, we are encouraged by the progression free survival of 3/8 patients in this dose escalation trial.

Citation Format: Thomas C. Chen, Naveed Wagle, David Peereboom, Lynne Taylor. Intranasal brain delivery using NEO100 for recurrent 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 CT064.
Abstract CT148: Small molecule immune-modulator icaritin: Safety, durable survival and inflammation-immune biomarkers in advanced hepatocellular carcinoma
Authors: Qin Shu-Kui , Li Qing , Xu Jianmin , Liang Jun , Cheng Ying , Li Shu , Zheng Limin , Ye Bin , Meng Kun , Sun Yan
Published: August 2019

Objectives: Hepatocellular carcinoma (HCC) was characterized with high heterogeneity and immune “tolerogenic”. Despite of immune checkpoint inhibitors have demonstrated promising results with improved overall survival. Novel small molecule-based immune therapies with high safety profiles might be particularly needed for advanced HCC patients. The purpose of this Phase II study is to explore the safety, clinical activities and treatment associated inflammation-immune biomarkers of icaritin in advanced HCC.

Methods: Major eligibility criteria include histologically confirmed unresectable, HCC patients with Child-Pugh Class A or B liver function. Total of 70 advanced HCC patients were enrolled and administrated with 600 mg b.i.d. Primary endpoints were TTP, and secondary endpoints were safety, OS, and DCR. Local disease control was defined as no progressive disease (PD) by RECIST. Kaplan-Meier analysis was utilized for OS assessment. Inflammation-Immune biomarkers NLR, PLR, cytokines, baseline expression of checkpoint/immune gene panel including PD-L1/L2, TIM3, IDO, HLA were evaluated by Nanostring and NGS technologies, retrospectively.

Results: There was no ≥ grade III drug related AE observed in all enrolled 70 advanced HCC patients. Objective response evaluation in per-protocol population showed PR (1.6%), SD (32.8%) and PD (59.0%) and median overall survival (OS) 254day (95% CI, 172-296). DCR was achieved 34.4% (95% CI, 22.7-47.7%); Median OS for the PD-L1-positive (n=9) and negative (n=24) subgroups were 389 (95% CI, 80-522) vs. 286.5days (95%CI, 135-482), for IL-6-advantage (n=28) and disadvantage (n=16) subgroups were 328.5 (95% CI, 277-566) vs. 168 days (95% CI, 135-482), for IL-8 low (n=22) and high (n=22) subgroups were 475.5(95% CI, 309-NA) vs. 175days (95% CI, 135-295), respectively. Overall survival was significantly associated with patient genetic profiles such as neo-antigen, bacterial and viral DNA inserts.

Conclusion: Icaritin has demonstrated its favorable clinical safety and preliminary immune-modulation efficacy. Potential baseline immune biomarkers to predict the improved OS were suggested including immune cell PD-L1 expression, low level peripheral IL-8, PLR, neoantigen and infection-associated genetic variations. Both safety and immune-response efficacy are warranted for the further Phase III validation.

Citation Format: Shu-Kui Qin, Qing Li, Jianmin Xu, Jun Liang, Ying Cheng, Shu Li, Limin Zheng, Bin Ye, Kun Meng, Yan Sun. Small molecule immune-modulator icaritin: Safety, durable survival and inflammation-immune biomarkers in advanced hepatocellular carcinoma [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 CT148.
Abstract LB-014: Antibody targeting soluble NKG2D ligand sMIC sensitizes metastatic prostate tumor and other MIC+ tumors to PD1/PD-L1 blockade therapy in pre-clinical models
Authors: Wu Jennifer D. , Zhang Jinyu , Larrocha Pablo , Dhar Payal
Published: August 2019

Targeting programmed cell death-1 (PD-1) has produced a survival benefit for patients diagnosed with many types of malignancies. However, the clinical survival benefit in metastatic prostate cancer is only marginal to-date. Currently, the understanding of why metastatic prostate cancer is resistant to PD-1 blockade therapy remains to be defined. Among the many factors that negatively impact response to PD-1 blockade treatment, tumor-derived soluble NKG2D ligands have been reported to hinder the clinical response to immune checkpoint blockade in melanoma patients. Metastatic prostate cancer cells release high levels of the circulating soluble NKG2D ligand, soluble MHC I Chain related molecule (sMIC), a molecule that is absent in classical rodent tumor model. Here, we tested the hypothesis that targeting sMIC sensitizes metastatic prostate tumors to PD-1 blockade therapy. Using a well-described double transgenic TRAMP/MIC mouse model that closely resembles the onco-immune dynamics of human metastatic prostate cancer, we demonstrate that an antibody targeting sMIC significantly enhances the response of prostate tumors to PD-1/PD-L1 blockade therapy. Mechanistically, targeting sMIC enriches NK cell and CD8 T cell infiltration into tumors, equips CD8 T cells with enhanced NKG2D and CD28 dual co-stimulation, and increases the expression of PD-L1+ on prostate tumor cells. Combination of the antibody targeting sMIC and a PD-1/PD-L1 blockade cooperatively augments intra-tumoral NK and antigen-specific CD8 T cell anti-tumor immunity. These findings were validated in syngeneic prostate tumor transplant models and other models of other cancer types. Our pre-clinical study provides proof-of-concept and uncovered novel mechanisms of a viable combination immunotherapy for metastatic prostate cancer and other MIC+ cancers.

Citation Format: Jennifer D. Wu, Jinyu Zhang, Pablo Larrocha, Payal Dhar. Antibody targeting soluble NKG2D ligand sMIC sensitizes metastatic prostate tumor and other MIC+ tumors to PD1/PD-L1 blockade therapy in pre-clinical models [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 LB-014.
Abstract LB-017: Combining tumor mutational burden, CD8+ T-cell abundance and PD1 mRNA expression accurately predicts response to anti-PD1/PDL1 therapy across cancers
Authors: Lee Joo Sang , Ruppin Eytan
Published: August 2019

Introduction Anti-PD1/PDL1 therapies provide significant survival benefits in many cancers, but the efficacy of these treatments varies considerably across different cancer types. Identifying the underlying variables associated with the cancer type specific response remains an important open research challenge. Here, we systematically evaluate a multitude of variables to determine the key factors that can accurately predict the response to anti-PD1/PDL1 therapy across different cancer types.

Approach To this end we analyzed whole exome sequencing (WES) and RNA sequencing (RNAseq) of 10k patients from the Cancer Genome Atlas (TCGA) and the overall response rate (ORR) to anti-PD1/PDL1 therapy of 21 cancer types obtained from previous clinical trials. We considered more than 30 different variables of three distinct classes: those associated with (a) tumor neoantigen landscape, (b) tumor microenvironment and inflammation, and (c) the checkpoint inhibitor targets. We evaluated the performance of each of these variables and their combinations in predicting the ORR to anti-PD1/PDL1 therapy. Prediction accuracy was quantified with Spearman correlation and explained variance quantified through a standard leave-one-out cross-validation.

Results Our analysis shows CD8+ T-cell abundance is the most predictive of the response to anti-PD1/PDL1 therapy across cancer types, followed by the tumor mutational burden and the fraction of samples with high PD1 gene expression in each cancer type. Notably these top three predictors cover the three distinct classes considered, and their combination is highly predictive of the ORR to anti-PD1/PDL1 therapy (Spearman R=0.9, P<1E-7), explaining more than 80% of the variance observed across different tumor types.

Conclusions and Relevance This is the first systematic evaluation of the different variables associated with PD1/PDL1 therapy response across different tumor types. Our findings show that, overall cancer types, three key variables can explain most of the observed cross-cancer response variability, but that their relative explanatory roles may vary in specific cancer types.

Citation Format: Joo Sang Lee, Eytan Ruppin. Combining tumor mutational burden, CD8+ T-cell abundance and PD1 mRNA expression accurately predicts response to anti-PD1/PDL1 therapy across cancers [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 LB-017.
Abstract LB-024: Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer
Authors: Jusu Sylvester , Presley John , Jean-Claude Bertrand , Stochaj Ursula , Kremer Richard
Published: August 2019

Purpose : The hormonally active form of vitamin D [1,25(OH)2D3], has several antitumor effects including antiproliferative, prodifferentiative and proapoptotic functions in tissues expressing and binding the vitamin D receptor. 1,25(OH)2D3 is synthesized from its precursor 25-hydroxyvitamin D [25(OH)D] via the catalytic action of the mitochondria cytochrome P450 enzyme 25(OH)D-1α-hydroxylase (CYP27B1). In the current study we investigated the role of CYP27B1 ablation on vitamin D signaling using the mouse mammary tumor virus promoter-driven polyoma middle T oncoprotein (MMTV-PyMT) mouse, an oncogene-driven model of highly aggressive spontaneous mammary tumors that closely mimics the estrogen receptor negative breast cancer in human disease.

Methods: Tumors from animals were digested and primary cells were obtained from both CYP27B1 ablated AOH93Cre+ cells and wild type MT1107 Cre- control cells. The cells were next grown in complete DMEM medium, transfected with either a GFP-tagged human VDR or RXRα and treated with 1,25(OH)2D3 (10−7 mol/L) or 25(OH)D3 (10−7 mol/L) before data acquisition.

Results: Using live and fixed cell fluorescence imaging, in situ proximity ligation assay and immunoblotting techniques, we show that the nuclear localization of both endogenous and exogenously tagged VDR and RXR were impaired in CYP27B1 ablated cells when compared to non-ablated cells. Furthermore, the intranuclear shuttling of VDR and its interaction with RXR were significantly reduced in CYP27B1 ablated cells as shown by both Florescence Recovery After Photobleaching and Fluorescence Resonance Energy Transfer techniques. Our results also show that CYP27B1 ablation disrupts VDR function by significantly impairing its nuclear localization, intranuclear transport, interaction with adaptor proteins, hVDR/hRXR, and this correlated with impaired binding to DNA and chromatin. Lastly, we demonstrate impaired induction of 53BP1, p65 and γ-H2AX DNA double-strand breaks in the CYP27B1 ablated cells compared to non-ablated cells.

Conclusion: These results suggest that ablation of CYP27B1 results in dysregulation in vitamin D signaling which may impair its anticancer functions.

Citation Format: Sylvester Jusu, John Presley, Bertrand Jean-Claude, Ursula Stochaj, Richard Kremer. Inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1): evidence for impaired vitamin D signaling in an MMTV-PYMT mouse model of breast cancer [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 LB-024.
Abstract LB-034: Myc instructs and maintains pancreatic adenocarcinoma phenotype
Authors: Sodir Nicole M. , Kortlever Roderik M. , Barthet Valentin J.A. , Pellegrinet Luca , Campos Tania , Kupczak Steven , Soucek Laura , Arends Mark J. , Littlewood Trevor D. , Evan Gerard I.
Published: August 2019

Pancreatic ductal adenocarcinoma (PDAC) sporadically develops from pre-cancerous pancreatic intraepithelial neoplasia (PanIN). It has a very poor prognosis and lacks effective therapies. Expression of the pleiotropic transcription factor Myc is deregulated in the majority of human cancers, including PDAC. Using a rapidly and reversibly switchable genetic mouse model to toggle Myc activity, we show that Myc drives immediate transition of indolent KRasG12D-induced PanINs into aggressive pancreatic adenocarcinomas that share all the signature features of spontaneous human PDAC. Myc induces a distinct set of instructive signals, some hitherto unknown, that together orchestrate coordinated changes in the multiple, distinct stromal and inflammatory cell types that generate the characteristic PDAC stroma. We also demonstrate that continued Myc activity is required for PDAC maintenance - Myc deactivation immediately triggers meticulous disassembly of PDAC, including its attendant stroma. Thus, both the formation and deconstruction of the complex PDAC phenotype can be mediated by a single, reversible molecular switch. We have also identified a previously unknown Myc-dependent GAS6-AXL pathway that is required for stromal changes characteristic of PDAC. Using the same pre-clinical mouse PDAC model, we show that TP-0903, a specific inhibitor of AXL kinase, profoundly blocks Myc-induced influx of neutrophils into PanINs and B220+ lymphoid cells into the PanIN periphery. This significantly retards Myc-driven stellate cell activation and proliferation and impedes PDAC growth. Hence, the GAS6-AXL-mediated signaling pathway is a promising therapeutic target for pancreatic ductal adenocarcinoma. TP-0903 is currently in a Phase I/Ib clinical study in in patients with solid tumors, including PDAC.

Citation Format: Nicole M. Sodir, Roderik M. Kortlever, Valentin J.A. Barthet, Luca Pellegrinet, Tania Campos, Steven Kupczak, Laura Soucek, Mark J. Arends, Trevor D. Littlewood, Gerard I. Evan. Myc instructs and maintains pancreatic adenocarcinoma phenotype [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 LB-034.
Abstract LB-036: Tetraspanin 5 expressing macrophages promote mammary tumor growth
Authors: Moamin Mohammed Ridha , Monk Peter , Cox Angela , Ottewell Penelope , Saint-Pol Julien , Rubinstein Eric , Muthana Munitta
Published: August 2019

Rationale: Tetraspanins (TSPANs) have emerged as key players in regulating tumor cell migration, invasion, and metastasis. Whilst we know TSPANs are expressed by macrophages very little is known regarding their role in Tumor Associated Macrophages (TAMs). We have shown that all 33 TSPAN members are differentially expressed by macrophages; however, following exposure to tumor-derived factors we see a significant increase in TSPAN5. We hypothesize that TSPAN5 plays a role in supporting the pro-tumor activities of TAMs and targeting tetraspanins in cancer may be a novel therapeutic approach.

Objectives: To determine TSPAN5 expression by macrophages in vitro and in situ in human breast cancer tissue and investigate the role of TSPAN5 in regulating the pro-tumor phenotype of TAMs.

Methodology: Expression of TSPAN5 was confirmed at the gene and protein level by qPCR, flow cytometry, western blotting, immunocytochemistry and by immunohistochemistry in formalin fixed breast cancer tissue. Accell siRNA knockdown was performed to determine the role of TSPAN5 in macrophage phenotype and function. NextSeq500 was performed to identify how TSPAN5 influences gene expression in BMDMs prepared from C57Bl/6 wild-type (WT) and TSPAN5 knockout (KO) mice. The role of TSPAN5 in a mammary carcinoma model in C57BL/6 WT and TSPAN5KO mice was also determined.

Findings: TSPAN5 was up-regulated by tumor-conditioned macrophages both in vitro and in vivo. This expression was also evident in TAMs located in patient derived breast cancer tissue (n=30). Interestingly, 64% of TAMs expressed TSPAN5 and these cells were the main source of TSPAN5 in human breast tumors (77%). Knockdown of TSPAN5 promoted a pro-inflammatory macrophage phenotype and NextSeq500 (n=3) revealed significant changes in genes involved in cell adhesion (Fn1, Cdh11, Lamb1, Cyr61, Postn and Wisp2) and immune regulation (PTGS2, GPX8 and Crip2) in BMDMs from TSPAN5KO mice compared to WT mice. Genetic and antibody ablation of TSPAN5 in C57BL/6 significantly reduced primary EO771 tumors and enhanced the survival of tumor bearing mice. Interestingly, clodronate depletion of macrophages in TSPAN5KO mice promoted mammary tumor growth.

Conclusion: Our study demonstrates for the first-time expression of TSPAN5 by TAMs in breast cancers and the pro-tumor properties of TSPAN5 in primary and metastatic mammary tumor growth.

Citation Format: Mohammed Ridha Moamin, Peter Monk, Angela Cox, Penelope Ottewell, Julien Saint-Pol, Eric Rubinstein, Munitta Muthana. Tetraspanin 5 expressing macrophages promote mammary tumor growth [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 LB-036.
Abstract LB-038: Development and characterization of cancer cell line exosomes as reference standards in cancer research
Authors: Paul Siddhartha , Mahashetty Nanda , Wells Joy , Yin Dezhong , Tian Fang
Published: August 2019

Exosomes are subcellular particles 50-200 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases, including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor-derived exosomes have been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for the isolation of large quantities of highly purified, ready to use exosomes from cell culture supernatant. Exosomes from a variety of cancer cell lines representing the most prevalent cancer types, including prostate (LNCaP), colorectal (HCT116), breast (MDA-MB-231, MCF-7), lung (A549), cervical (HeLa), and glioblastoma (U87-MG) were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method generated a high yield (>1×1010 exosomes/mL) and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different characteristic exosomal proteins including tetraspanin, which was confirmed through western blot and PCR. Isolated exosomes had a median size of 102 nm through nanoparticle tracking analysis. Western blot data revealed cell line specific protein marker expression of CD63, CD81, flotillin 1, Alix and TSG101 protein. We assessed the functionality of these purified exosomes through cellular uptake, migration assay, and anchorage independent growth assay in this study. We also evaluated exosomal RNA from these purified exosomes for the presence of common mutations associated with cancer through PCR. Here we report that exosomes isolated by our method not only had high exosome yield and quality, but also retained relevant functionality and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development.

Citation Format: Siddhartha Paul, Nanda Mahashetty, Joy Wells, Dezhong Yin, Fang Tian. Development and characterization of cancer cell line exosomes as reference standards in cancer research [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 LB-038.