bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2924: Evaluation of the pan-FGFR inhibitor AZD4547 with radiation in non-small cell lung cancer
Authors: Miller Margot , Fisher Michael , Senthilkumar Gopika , Kaushik Saakshi , Able Lindsey , Brennan Sean , Iyer Gopal , Kimple Randall , Baschnagel Andrew M.
Published: August 2019

Objective: The fibroblast growth factor receptors (FGFR) are commonly altered in non-small cell lung cancer (NSCLC), including high level of amplification of FGFR1 in lung squamous cell carcinoma. FGFR signaling may play a role in the response to radiation. We investigated the radiosensitizing effects of the selective tyrosine kinase inhibitor AZD4547 in a NSCLC model using cell lines and tumor xenografts.

Methods: A panel of six NSCLC cell lines were screened for FGFR1/2 DNA amplification, RNA expression, and protein expression. All cells were assessed for in vitro response to AZD4547. Immunoblots were used to examine the effect of AZD4547 on downstream signaling proteins. Apoptosis was measured by Annexin V staining and autophagy with acridine orange assay. Radiation clonogenic survival assays and xenograft growth delays experiments were performed to investigate radiosensitization. In vivo mechanistic studies were conducted using immunohistochemistry.

Results: Cell lines demonstrated varying degree of FGFR1/2 RNA and protein expression. In sensitive cell lines, AZD4547 inhibited p-MAPK in a time dependent manner. In vitro clonogenic survival assays showed robust radiosensitivity with AZD4547 in three out of six NSCLC cell lines. All three cell lines overexpressed FGFR1 and two of the cells had high FGFR1 copy number. There was no radiosensitization seen in an immortalized normal human bronchial epithelial cell line. A significant increase in autophagy and apoptosis was observed with combined radiation and AZD4547. Significant tumor growth delay was observed with the administration of radiation and AZD4547 compared to radiation or drug alone in two NSCLC xenograft tumor models. IHC analysis revealed modulation of FGFR-downstream signaling (p-Erk and p-S6) and proliferative (Ki67) and apoptotic markers (Cleaved Caspace 3).

Conclusion: These findings suggest that AZD4547 can augment the response of radiation in NSCLC model systems. FGFR1 and FGFR2 expression may be a potential targets for radiosensitization in NSCLC. Additional studies are underway to understand the mechanism of radiosensitization.

Citation Format: Margot Miller, Michael Fisher, Gopika Senthilkumar, Saakshi Kaushik, Lindsey Able, Sean Brennan, Gopal Iyer, Randall Kimple, Andrew M. Baschnagel. Evaluation of the pan-FGFR inhibitor AZD4547 with radiation in non-small cell 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 2924.
Abstract 2934: AKT mutant allele-specific activation dictates pharmacologic sensitivities
Authors: Bhattarai Tripti Shrestha , Shamu Tambudzai , Patel Swati , Gorelick Alexander , Chang Matthew T. , Gavrila Elena I. , Gao JianJong , Donoghue Mark T. , Gao Paul S. , Soumerai Tara , Abida Wassim , Smyth Lilian M. , Hyman David M. , Solit David B. , Taylor Barry S.
Published: August 2019

The objective of this study is to explore the biochemical characteristics and therapeutic sensitivity profiles of novel, non-E17K, less frequent AKT1 mutations, and thereby expand the biomarker of sensitivity to AKT inhibition in molecularly defined cancer patients. AKT is a critical signaling node that translates phosphoinositide 3-kinase (PI3K) pathway stimulation into diverse cellular effects. Gain of function AKT1 mutations arise in diverse human cancers, of which E17K is the most common. Presence of AKT1 E17K mutation renders these tumors susceptible to AKT inhibition. Nevertheless, the long tail of potentially activating mutations in AKT is largely uncharacterized, thereby limiting our ability to act clinically in prospectively sequenced advanced cancer patients. We performed a population-scale candidate driver mutation discovery analysis in AKT1, AKT2 and AKT3 in a cohort of 41,075 retrospectively and prospectively sequenced primary and metastatic cancers, and explored both their functional and biological impact as well as their therapeutic sensitivity. Our results demonstrated that some, but not all of the identified AKT missense mutations activated PI3K signaling in a growth factor-independent manner, and sensitized tumor cells to diverse AKT inhibitors. By contrast, we discovered a different class of small in-frame paralogous AKT duplication mutants that induced distinctive structural changes, leading to a far greater degree of membrane affinity, AKT activation, pathway dependence, and hyper-sensitivity to ATP-competitive AKT inhibitors, while conferring resistance to allosteric AKT inhibitors. Leveraging a co-clinical trial framework, we are now enrolling patients on the basis of these mutations in a basket study involving AKT alterations. One such case was that of a castration-resistant metastatic prostate cancer patient who harbored AKT2 duplication mutant, and subsequently responded to AKT inhibition. Collectively, our data indicate that the degree and mechanism of activation of oncogenic AKT mutants vary, thereby dictating allele-specific pharmacological sensitivities to AKT inhibition.

Citation Format: Tripti Shrestha Bhattarai, Tambudzai Shamu, Swati Patel, Alexander Gorelick, Matthew T. Chang, Elena I. Gavrila, JianJong Gao, Mark T. Donoghue, Paul S. Gao, Tara Soumerai, Wassim Abida, Lilian M. Smyth, David M. Hyman, David B. Solit, Barry S. Taylor. AKT mutant allele-specific activation dictates pharmacologic sensitivities [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 2934.
Abstract 2943: Severe hepatotoxicity of mithramycin therapy caused by altering expression of hepatocellular bile transporters
Authors: Sissung Tristan M. , Huang Phoebe A. , Hauke Ralph , McCrea Edel , Peer Cody J. , Barbier Roberto H. , Strope Jonathan D. , Ley Ariel M. , Zhang Mary , Hong Julie A. , Venzon David , Jackson Jonathan P. , Brouwer Kenneth R. , Grohar Patrick , Glod John , Widemann Brigitte C. , Heller Theo , Schrump David S. , Figg William D.
Published: August 2019

Mithramycin has shown significant preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with inter-individual variation in genes involved in bile disposition: ABCB4(MDR3) rs2302387 and ABCB11(BSEP) rs4668115 variants reduce transporter expression (P<0.05) and were associated with ≥Grade 3 liver function test (LFT) elevations developing 24 hours after the third infusion of mithramycin (25mcg/kg, 6hr/infusion, qdx7, every 28 days;P<0.0040). A similar relationship was observed in a pediatric cohort genotyped for ABCB11. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, NTCP, OSTα/β) in several cell lines (Huh7, HepaRG, HepaRG BSEP (-/-)) and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P<0.0001), and mithramycin inhibited CDCA- and GW4046-induced FXR-GAL4 luciferase reporter activity (P<0.001). Mithramycin promoted GCDC-induced cytotoxicity in cells lacking the BSEP transporter and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P<0.01). Mithramycin is an FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low BSEP function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses.

Citation Format: Tristan M. Sissung, Phoebe A. Huang, Ralph Hauke, Edel McCrea, Cody J. Peer, Roberto H. Barbier, Jonathan D. Strope, Ariel M. Ley, Mary Zhang, Julie A. Hong, David Venzon, Jonathan P. Jackson, Kenneth R. Brouwer, Patrick Grohar, John Glod, Brigitte C. Widemann, Theo Heller, David S. Schrump, William D. Figg. Severe hepatotoxicity of mithramycin therapy caused by altering expression of hepatocellular bile transporters [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 2943.
Abstract 2967: Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells
Authors: Montoya Justin J. , Turnidge Megan A. , Wai Daniel H. , Lee David W. , Patel Apruvi , Gokhale Vijay , Hurley Laurence J. , Azorsa David O.
Published: August 2019

Acute Myeloid Leukemia (AML) is a malignancy of myeloid precursor cells that can occur when genomic changes alter expression of key genes, causing cells to resume an undifferentiated state and proliferate. Ongoing efforts have focused on developing therapies that specifically target the protein products of aberrantly expressed genes. However, many of the identified proteins are difficult to target because of structural challenges, protein overexpression, or mutations that confer resistance to therapy. A type of therapy that circumvents these issues is the use of small molecules that stabilize DNA secondary structures called G-quadruplexes, which are present in the promoters of many potential oncogenes, and have regulatory roles in their transcription. This study analyzes the activity of G-quadruplex stabilizing small molecule GQC-05 that has been shown to down-regulate MYC, which is commonly misregulated in AML. Treatment of MYC-expressing AML cell lines KG-1a, CMK and TF-1 with GQC-05 resulted in decreased expression of MYC mRNA and protein, with the effect more pronounced in KG-1a cells. GQC-05 treatment of the AML cells decreased cell viability, while increasing apoptosis and DNA damage. Combinational drug screening was performed to identify compounds that potentiated the anti-proliferative effects of GQC-05. Results from the screen identified the BCL-2 inhibitor Navitoclax as a compound that potentiated GQC-05 activity. Co-treatment with GQC-05 and Navitoclax showed synergistic effect on cell viability of AML cells as determined by Chou-Talalay analysis. Furthermore, co-treatment with Navitoclax did not affect the downregulation of MYC by GQC-05, but did increase apoptosis and DNA damage leading to rapid cytotoxicity. These results indicate that the G-quadruplex stabilizing small molecule GQC-05 may function more as a DNA damaging agent in AML cells and that combining GQC-05 with a Bcl-2/Bcl-XLinhibitor such as Navitoclax can result in increased cytotoxic activity.

Citation Format: Justin J. Montoya, Megan A. Turnidge, Daniel H. Wai, David W. Lee, Apruvi Patel, Vijay Gokhale, Laurence J. Hurley, David O. Azorsa. Activity of G-quadruplex stabilizing small molecule that downregulates MYC and synergizes with Navitoclax in acute myeloid leukemia cells [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 2967.
Abstract 2980: The role of USP7 and USP7 inhibitor in HER2+ breast cancer treatment
Authors: Yu Jia , Qin Bo , Boughey Judy , Goetz Matthew , Wang Liewei
Published: August 2019

INTRODUCTION: Breast cancer is the most common invasive cancer and the second leading cause of cancer death in women. Approximately 20% of breast cancer tumors overexpress human epidermal growth factor receptor 2 (HER2), which is associated with an aggressive clinical phenotype and with a poorer prognosis. The development of trastuzumab, an immunoglobulin G monoclonal antibody directed against HER2, has changed the treatment paradigm for this disease. However, approximately 15-20% still does not respond to anti-HER2 therapy. Furthermore, despite advances in the management of metastatic HER2-positive breast cancer, response rate varies greatly, with most patients eventually succumbing to their disease. It is crucial to develop additional therapeutic strategies to improve outcomes for HER2 positive patients, especially those with tumors that do not respond to anti-HER2 therapy.

METHODS and RESULTS: Using HER2+ breast cancer cell lines, we found that an USP7 inhibitor, P5091, dramatically sensitized cancer cells to HER2 inhibitor, trastuzumab treatment. To confirm it in vivo, we tested the drug combination in BT474 xenografts and found P5091 and trastuzumab synergistically suppressed tumor growth. Furthermore, using trastuzumab resistant HER2+ breast cancer cell line, we found that USP7 inhibitor, P5091, can dramatically re-sensitize trastuzumab-resistant BT474 cell line to trastuzumab treatment both in vitro and in vivo. More importantly, using patient derived xenograft (PDX) HER2+ models, we confirmed the synergistic combination effect of P5091 and trastuzumab. Mechanistically, USP7 inhibitor, P5091 led to significantly increased degradation of all four HER family members (HER1-4). When we knocked down USP7 by RNA interference or small hairpin RNA (shRNA), we observed similar substantial decrease in HER family member proteins.

CONCLUSIONS: We have demonstrated the feasibility of using USP7 inhibitor P5091 to sensitize HER2+ breast cancers to trastuzumab treatment. These results provide a rationale for the use of combination therapy of USP7 inhibitors and HER2 inhibitors to treat HER2+ breast cancer. This combination therapy might also be effective in other cancers with upregulated HER signaling, such as GI cancer, lung cancer or ovarian cancer.

Citation Format: Jia Yu, Bo Qin, Judy Boughey, Matthew Goetz, Liewei Wang. The role of USP7 and USP7 inhibitor in HER2+ breast cancer treatment [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 2980.
Abstract 2983: LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral cancer
Authors: Miwa Takashi , Yamada Shinji , Kaneko Mika K. , Furusawa Yoshikazu , Fukui Masato , Kato Yukinari
Published: August 2019

Background

Podoplanin (PDPN) is a specific ligand of C-type lectin-like recepotor-2 (CLEC-2), and is involved in cancer metastasis. The physiological PDPN function has been discussed in many normal tissues, including lymphatic endothelial cells, pulmonary type I alveolar cells, and renal podocytes. Herein, we aimed to produce cancer-specific anti-PDPN monoclonal antibodies (mAbs). Furthermore, we investigated whether the reactivity of cancer-specific anti-PDPN mAbs might be a prognostic marker of oral cancer.

Materials and methods

We immunized mice with LN229/PDPN cells, and screened mAbs, which react with PDPN-expressing cancer cell lines, such as glioblastoma cell lines and lung squamous cell carcinoma cell lines and do not react with PDPN-expressing normal cells, including primary lymphatic endothelial cells and renal epithelial cell lines in flow cytometry. Furthermore, we investigated the reactivity of anti-PDPN mAbs by immunohistochemistry against oral cancers. Anti-tumor activities by mouse-human chimeric LpMab-23 (chLpMab-23) were examined using mouse xenograft models of oral cancer cells. Finally, we analyzed the association between the reactivity of anti-PDPN mAbs and clinical/pathological features of oral cancers.

Results

We established LpMab-23, a cancer-specific anti-PDPN mAb. LpMab-23 reacted with PDPN-expressing cancer cell lines in flow cytometry. In contrast, LpMab-23 recognized PDPN-expressing normal cells very weakly. LpMab-23 reacted only with PDPN-expressing cancer cells, not with lymphatic endothelial cells in oral cancer tissues by immunohistochemistry. The epitope mapping of anti-PDPN mAbs revealed that LpMab-23 recognized a cancer-specific glycopeptide including Thr55/Ser56. chLpMab-23 revealed high ADCC and anti-tumor activities against oral cancers. The Kaplan-Meier curves of the five-year new metastasis-free survival rate (nMFS) were significantly lower in LpMab-23-positive patients than in the patients with LpMab-23-negative ones.

Conclusions

A cancer-specific anti-PDPN mAb (LpMab-23) and its mouse-human chimeric mAb (chLpMab-23) were successfully established. LpMab-23 is advantageous in anti-tumor activities. LpMab-23-positive cases could be a useful predictor of poor prognosis for oral cancer.

Citation Format: Takashi Miwa, Shinji Yamada, Mika K. Kaneko, Yoshikazu Furusawa, Masato Fukui, Yukinari Kato. LpMab-23: an anti-podoplanin cancer-specific antibody for a prognostic marker of oral 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 2983.
Abstract 2997: 15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1
Authors: Satapathy Shakti R. , Mehdawi Lubna M. , Olsson Roger , Sjolander Anita
Published: August 2019

Prostaglandin E2 (PGE2) is a crucial regulator of colon cancer (CC), an inflammation associated cancer. Tumor suppressor 15-hydroxyprostglandin dehydrogenase (15-PGDH) is a PGE2 degrading enzyme but is often downregulated in CC. Interestingly, CC-patients with high levels of Cysteinyl leukotriene receptor 2 (CysLT2R) exhibit good prognoses. Previously, we have reported differentiation promoting role of 15-PGDH in CC cells when induced by LTC4, the CysLT2R ligand. However the mechanism is unclear. Hedgehog (Hh) signalling is involved in embryogenesis and development but hyper activated in colon carcinogenesis. LTC4-induced activation of 15-PGDH downregulated both mRNA and protein expression of GLI1 in HT-29 and Caco-2 colon cancer cells. Moreover, absence of Gli1 significantly reduced the differentiation promoting ability of 15-PGDH, with no change in the differentiation markers Sucrase isomaltase, Mucin-2 and Protocadherin-24. In addition, no noticeable change in the mRNA expression of WNT-specific target AXIN-2 coupled with restoration of the proliferation marker cMYC was observed. In HT-29-derived colonospheres depletion of mRNA expression of CC-CSC markers ALDH1A1 and DCLK1 remained regulated by Gli1. Elevation in Mucin-2 expression in zebrafish xenograft with HT-29 was also noted to be Gli1 dependent. GLI1 and MUCIN-2 mRNA expression in normal mucosa and matched tumor in colon cancer patient also exhibited higher co-relation along with concomitant elevation in DCLK1 expression in tumor in compared to normal mucosa. Tumor microarray (TMA) with low expression of CysLT2R and 15-PGDH exhibited higher Gli1 expression with no trace of Mucin-2. Therefore, restoring the expression of 15-PGDH in CC through activation of CysLT2R signalling and targeting Hh-Gli signalling would open new therapeutic possibilities with minimal collateral damage to the normal tissue.

Citation Format: Shakti R. Satapathy, Lubna M. Mehdawi, Roger Olsson, Anita Sjolander. 15-hydroxyprostaglandin dehydrogenase induced differentiation in colon cancer cells is regulated via Gli1 [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 2997.
Abstract 2998: Co-targeting bulk tumor and CSCs in clinically translatable TNBC patient-derived xenografts via combination nanotherapy
Authors: Sulaiman Andrew , El-Sahli Sara , McGarry Sarah , Wang Lisheng , Gadde Suresh
Published: August 2019

Triple negative breast cancer (TNBC) accounts disproportionally for the majority of breast cancer related deaths throughout the world. This is largely attributed to lack of a specific therapy capable of targeting both bulk tumor mass and cancer stem cells (CSC) as well as appropriate tumor animal models to accurately evaluate treatment efficacy for the clinical translation. Thus, development of effective and clinically translatable targeted therapies for TNBC is an unmet medical need. In this report, we developed a hybrid nanoparticles-based co-delivery platform containing both paclitaxel and verteporfin (PV-NPs) to target TNBC patient-derived xenograft (PDX) tumor and CSCs. MRI and IVIS imaging were performed on mice containing PDX tumors to assess tumor vascularity and selective accumulation of NPs. NF-kB, Wnt and YAP activities were measured by reporter assays. Mice bearing TNBC PDX tumor were treated with PV-NPs and controls; and tumors progression and CSC subpopulations were analyzed. Our MRI imaging studies indicated high vascularization of PDX tumors. IVIS imaging showed selective accumulation of NPs in PDX tumors. In comparison to control NPs and free-drug combination, PV-NPs significantly retarded tumor growth of TNBC PDX. PV-NPs simultaneously repressed NF-kB, Wnt and YAP that have been shown to be crucial for cancer growth, CSC development and tumorigenesis. In conclusion, NPs containing two clinically used drugs concurrently inhibited NF-kB, Wnt and YAP pathways and exhibited synergic effects on killing TNBC bulk tumor and CSCs. This combination nanotherapy evaluated with a PDX model may lead to an effective treatment of TNBC patients.

Note: This abstract was not presented at the meeting.

Citation Format: Andrew Sulaiman, Sara El-Sahli, Sarah McGarry, Lisheng Wang, Suresh Gadde. Co-targeting bulk tumor and CSCs in clinically translatable TNBC patient-derived xenografts via combination nanotherapy [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 2998.
Abstract 3001: Isolation of large and diverse monoclonal antibody panels against difficult targets in oncology
Authors: Rucker Joseph
Published: August 2019

Although monoclonal antibodies (MAbs) are a well-established treatment approach in oncology, multipass membrane proteins are largely inaccessible as targets for MAb discovery due to their poor expression, membrane-dependent structure, small extracellular regions, and high sequence conservation between humans and rodents. We have developed a platform, MPS Antibody Discovery, that specifically addresses these challenges. We will present case studies of our successful isolation of large and diverse panels of MAbs, including functional (agonist/antagonist) MAbs, against oncology targets, including claudins, GLUT4, CXCR5, CCR7, and other chemokine receptors. Several key features of our MPS platform enabled our ability to isolate MAb panels against these targets. Antigen engineering allowed us to increase the expression levels of the target protein. Immunizing divergent animal species with a combination of DNA and Lipoparticles (high-concentration membrane proteins) allowed us to obtain high-titer immune responses against the native form of the membrane protein. Performing phage display with Lipoparticles allowed us to isolate diverse panels of MAbs with a success rate >95%. Our technology offers the potential for discovering MAbs against difficult targets in cancer, including neoantigens.

Citation Format: Joseph Rucker. Isolation of large and diverse monoclonal antibody panels against difficult targets in oncology [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 3001.
Abstract 3022: Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft
Authors: Filippidou Natalia , Valianou Mathildi , Johnson Daniel L. , Bissler John J. , Astrinidis Aristotelis
Published: August 2019

Despite the identification of mTOR hyperactivation as the main biochemical defect downstream of TSC1/TSC2 inactivation in Lymphangioleiomyomatosis (LAM) and Tuberous Sclerosis Complex (TSC), and the approval of rapamycin and rapalogs for the treatment of the related lesions, these drugs are cytostatic and continued treatment is required for clinical benefit. The latter raises the possibility of acquired drug resistance over long-term use. To explore the mechanisms leading to rapamycin resistance in LAM/TSC, we xenografted SCID mice with ELT3 cells (Tsc2-null derived from an Eker rat uterine leiomyoma), explanted a tumor that was not responsive to rapamycin, and generated cell line ELT3-245. The cell growth of ELT3-245 is not inhibited by rapamycin in vitro. SCID mice inoculated with ELT3-245 form palpable tumors significantly faster, compared to ELT3 cells, respond very poorly to rapamycin at the initial stages of treatment, but soon become non-responsive. In addition, these cells form macroscopically visible metastases to the lungs of ELT3-245 tumor-bearing mice. We performed global gene expression profiling, comparing ELT3-245 to ELT3 cells, and found that the rapamycin-resistant cells have decreased expression of genes associated with epithelial cells and increased expression of genes associated with mesenchymal-like characteristics. Relative gene expression of Myc, Egfr, Akt2, Jun, and Ocln were assessed by RT-qPCR in ELT3 vs ELT3-245 after 24h of treatment with 20 nM rapamycin or DMSO. Important discrepancies include a 2.8-fold increased expression of Egfr under rapamycin treatment in ELT3 cells but not in ELT3-245; however, ELT3-245 had a 15-fold higher expression of Egfr prior to treatment, compared to ELT3. In addition, ELT3-245 cells strikingly increase expression of Myc when challenged with rapamycin, compared to ELT3. Similarly, Akt2 is increased considerably under rapamycin treatment in ELT3-245 and is already higher prior to treatment compared to ELT3. Finally, Ocln expression was at least 100-fold reduced in ELT3-245, compared to ELT3, both under rapamycin treatment and no-treatment conditions. These results suggest that Myc, Egfr, Akt2, and Ocln are among the genes highly relevant to acquired rapamycin resistance in this Tsc2-null rat leiomyoma cell line and point toward the pathways that need to be interfered with in order to overcome it.

Citation Format: Natalia Filippidou, Mathildi Valianou, Daniel L. Johnson, John J. Bissler, Aristotelis Astrinidis. Dissecting rapamycin resistance in a Tsc2-null rat leiomyoma cell line developed in a murine xenograft [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 3022.
Abstract 3028: STAT3 represses miR-145-5p to exacerbate HER3 expression for surviving EGFR-TKIs in lung cancers
Authors: Cheng Chun-Chia , Chiang Ya-Wen , Lim Ken-Hong , Chang Jungshan , Ho Ai-Sheng , Chang Yi-Fang
Published: August 2019

HER3 exerts resistance against epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we have demonstrated that EGFR induces HER3 overexpression and that contributes to the formation of cancer stem-like tumorspheres. However, the cellular mechanism of EGFR in regulating HER3 expression was obscure. We hypothesized that EGFR-downstream STAT3 participates in HER3 expression since STAT3 contributes to cancer stemness and surviving EGFR-TKIs. First, RNAseq was used to uncover the potential genes involving in formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines (HCC827, A549, H1975) were individually treated with a panel containing 172 compounds which targeting to stem cell-associated genes in order to searching potential agents against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used for investigating the molecular mechanism of STAT3 on regulating tumor progression and survival of lung cancers. We found that BBI608, a STAT3 inhibitor, was a potential therapeutic agent specifically reducing the cell viability of EGFR-positive lung cancers. Interestingly, the inhibitory effects caused by BBI608 were similar with that derived from YM155, an ILF3 inhibitor, both compounds reduced G9a-mediated HER3 expression. We, furthermore, demonstrated that STAT3 upregulated G9a to silence miR-145-5p for exacerbating HER3 expression in this study. In conclusion, this study figured out the potential cellular function of STAT3 in EGFR-positive lung cancers. We also evaluated that BBI608 was potential to eradicate EGFR-positive lung cancers and demonstrated that STAT3 regulated expression of HER3, indicating that STAT3 was a reliable therapeutic target against EGFR-TKI-resistant lung cancers.

Note: This abstract was not presented at the meeting.

Citation Format: Chun-Chia Cheng, Ya-Wen Chiang, Ken-Hong Lim, Jungshan Chang, Ai-Sheng Ho, Yi-Fang Chang. STAT3 represses miR-145-5p to exacerbate HER3 expression for surviving EGFR-TKIs in lung 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 3028.
Abstract 304: Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity
Authors: Arita Masashi , Watanabe Satoshi , Aoki Nobumasa , Takahashi Miho , Shoji Satoshi , Nozaki Koichiro , Ichikawa Kosuke , Kondo Rie , Kuwahara Shoji , Tanaka Junta , Koya Toshiyuki , Saito Akihiko , Kikuchi Toshiaki
Published: August 2019

Cisplatin is an anticancer drug widely used in the treatment of many cancers, including lung cancers. Although cisplatin causes various types of adverse events, the main dose-limiting toxicity of cisplatin is nephrotoxicity. Megalin is an endocytic receptor expressed at the apical membranes of proximal tubules. We previously demonstrated that cisplatin was reabsorbed through megalin and caused kidney injury. Cilastatin, an inhibitor of renal dehydropeptidase-I and used with imipenem, blocked the binding of cisplatin to megalin and reduced the nephrotoxicity induced by cisplatin. In the current study, we precisely evaluated the effect of cilastatin-mediated suppression of cisplatin nephrotoxicity to safely enhance the antitumor activity of cisplatin. BALB/c mice were administrated cisplatin with or without cilastatin. Tubular dilation or atrophy, brush border loss, tubular cell lysis and cast formation were observed in mice treated with cisplatin alone. However, these kidney injuries were decreased or disappeared in mice treated with cisplatin and cilastatin. Cilastatin also decreased the urinary levels of N-acetyl-β-D-glucosaminidase and neutrophil gelatinase-associated lipocalin, proximal tubular injury markers. Next, SCID mice were injected s.c. with A549, a human lung cancer cell line, and treated with cisplatin with or without cilastatin. Cilastatin did not affect the antitumor activity of cilastatin. Notably, A549 did not express megalin. Combined with cilastatin, the mice were successfully treated with 1.5 times dose of cisplatin with enhanced antitumor effects of cisplatin but without nephrotoxicity. In conclusion, cilastatin effectively suppressed nephrotoxicity of cisplatin by blocking the binding of cisplatin to megalin. These findings indicated that we could administer cisplatin into cancer patients without nephrotoxicity if we used cilastatin. Moreover, we might increase the dose of cisplatin and improve the outcome of cancer patients.

Citation Format: Masashi Arita, Satoshi Watanabe, Nobumasa Aoki, Miho Takahashi, Satoshi Shoji, Koichiro Nozaki, Kosuke Ichikawa, Rie Kondo, Shoji Kuwahara, Junta Tanaka, Toshiyuki Koya, Akihiko Saito, Toshiaki Kikuchi. Combination therapy of cisplatin with cilastatin enables to increase the dose of cisplatin for enhancing its antitumor effect by suppressing nephrotoxicity [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 304.
Abstract 3068: Synergistic effect of BCL-2 inhibitor APG-2575 and CDK9 inhibitor in acute myeloid leukemia and DLBCL preclinical tumor models
Authors: Fang Douglas D. , Tao Ran , Tang Qiuqiong , Gu Shoulai , Zhai Guoqin , Li Jiajun , Wang Qixin , Fang Xu , Li Na , Yang Dajun , Zhai Yifan
Published: August 2019

Effective treatments are urgently needed for elderly acute myeloid leukemia (AML) and relapsed or refractory diffuse large B-cell lymphoma (DLBCL) patients as only 10-25% of the population respond to standard therapies and resistance often occurred. Concurrent overexpression of anti-apoptotic BCL-2 gene and proto-oncogene MYC are frequently found in AML and DLBCL and this molecular characteristic is associated with poor prognosis of patients. CDK9 controls non-ribosomal transcription of genes including MYC and MCL-1. Dysregulation in the CDK9 pathway had been reported in AML and DLBCL, making it an attractive target for the combination with BCL-2 inhibitor to achieve more effective therapy.

To test the above hypothesis, in this study, we applied the combination of APG-2575 with a CDK9 inhibitor (alvocidib or dinaciclib) to preclinical tumor models. APG-2575 is a novel, orally bioavailable BH3-mimetic that selectively inhibits BCL-2, but not BCL-xL or MCL-1. After the treatment, synergistic effect of the combination treatment was demonstrated in mice bearing xenograft tumors of human AML or DLBCL. Specifically, in xenograft tumor models derived from human DLBCL cell line U2932, the treatment with APG-2575, alvocidib, or dinaciclib single agent for 22 days exhibited antitumor activity with T/C values of 20-50% (T/C value, average tumor volumes in treatment group vs. the vehicle control group). The combination treatment achieved substantial antitumor activity with T/C value of 5%. All animals in the combination groups showed either partial or complete tumor regression whereas none in the single arms.

Similar synergistic antitumor activity of the combination treatment was consistently observed in the myelodysplastic syndrome SKM-1 and AML OCI-AML-3 xenograft models. Taken together, our data suggest that APG-2575 and CDK9 inhibition combination has a potential therapeutic application in treatment of patients with AML and DLBCL.

Citation Format: Douglas D. Fang, Ran Tao, Qiuqiong Tang, Shoulai Gu, Guoqin Zhai, Jiajun Li, Qixin Wang, Xu Fang, Na Li, Dajun Yang, Yifan Zhai. Synergistic effect of BCL-2 inhibitor APG-2575 and CDK9 inhibitor in acute myeloid leukemia and DLBCL preclinical tumor 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 3068.
Abstract 3078: Tesetaxel, a novel, oral taxane, crosses intact blood-brain barrier (BBB) at therapeutically relevant concentrations
Authors: James Joyce , Tang Kevin , Wei Thomas
Published: August 2019

Background: Central nervous system (CNS) metastases in cancers such as breast and lung cancer are common and associated with poor outcomes. The taxanes paclitaxel (P) and docetaxel (D) do not significantly cross the BBB (brain to plasma exposure of only 1% and 8%, respectively), making them ineffective in treating CNS metastases. Tesetaxel (T) is a novel, orally administered taxane currently in a Phase 3 study in patients with advanced breast cancer (aBC; NCT03326674). Several properties make it unique, including: oral administration with a low pill burden; a long (~8-day) terminal plasma half-life in humans, allowing infrequent dosing; no history of hypersensitivity reactions; significant activity against chemo-resistant tumors; and, unlike P and D, minimal efflux by the P-glycoprotein pump, a key element of the BBB. The in vivo tissue distribution of tesetaxel was previously investigated in mice treated with 14C-T at 4 mg/kg and sacrificed between 1-168 hours. We now report 2 additional studies in dogs and monkeys. Methods: Dogs and monkeys were dosed at 0.6 mg/kg and 1 mg/kg of 14C-T, respectively, and tissue distribution was assessed at 336 hours. These doses equate to ~44% of the 27 mg/m2 dose in the ongoing Phase 3 study. In vivo radioactivity concentrations in the cerebrum were compared to plasma concentrations and mean tumor GI50 (concentration resulting in 50% tumor growth inhibition) determined by MTT assay in 23 tumor cell lines. Results: The absolute brain concentrations of T in dogs and monkeys far exceeded both the respective plasma concentrations and the tumor GI50 (Table 1). Conclusions: In dogs and monkeys, T brain concentrations far exceed its tumor GI50 for a sustained period when administered at therapeutically relevant concentrations. The inclusion criteria for the ongoing Phase 3 study of T in patients with aBC allows for CNS metastases.

Table 1:Tesetaxel CNS Levels Far Exceed Human GI50a in Dogs and Monkeys14C-T Radioactivity Concentration (ng eq./g or mL) bCerebrum Concentration/ Tumor GI50 cNCerebrum (Mean ± SD)Plasma (Mean ± SD)Cerebrum/ PlasmaDog d310.9 ± 4.00.9 ± 0.112x18xMonkey e36.5 ± 3.80.9 ± 0.27x11xaConcentration of drug required to inhibit growth by 50%b14 days after dosingcMean GI50 for T across 23 tumor cells lines = 0.6 ng/mLdSingle dose of 0.6 mg/kg (equivalent to 44% of a human dose of 27 mg/m2)eSingle dose of 1 mg/kg (equivalent to 44% of a human dose of 27 mg/m2)

Citation Format: Joyce James, Kevin Tang, Thomas Wei. Tesetaxel, a novel, oral taxane, crosses intact blood-brain barrier (BBB) at therapeutically relevant concentrations [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 3078.
Abstract 3091: Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions
Authors: Sharma Priyanka , Diergaarde Brenda , Ferrone Soldano , Kirkwood John M. , Whiteside Theresa L.
Published: August 2019

Background: Exosomes are a subset of small (30-150nm) extracellular vesicles (EVs) of the endocytic origin that mediate intercellular communication in health and disease. Exosomes are actively produced by tumor cells, circulate freely in body fluids and carry messages that alter functions of normal cells in the tumor microenvironment (TME), including immune cell functions. The ability of melanoma exosomes to reprogram immune cell functions might contribute to disease progression.

Methods: To evaluate effects of melanoma cell-derived exosomes (MTEX) on human immune cell subsets, we isolated exosomes from plasma of 12 patients with melanoma and separated MTEX from non-MTEX by immune capture using anti-CSPG4 mAb on beads. These exosome fractions, and exosomes from plasma of 6 normal donors used as controls (NC), were studied by on-bead flow cytometry to quantify their surface protein profiles and in co-incubation assays with immune cell subsets for functional attributes. Phenotypic and functional profiles of MTEX and non-MTEX were compared.

Results & Conclusion: MTEX were enriched (p<0.005)( in FasL and TRAIL, induced apoptosis of CD8+T cells, down-regulated CD69 expression levels on T cells and inhibited their proliferation (all p<0.0005). MTEX also interfered with NK cell functions (p<0.001). Non-MTEX were enriched (P<0.005) in co-stimulatory proteins, CD40L, OX40L, OX40 and, similar

to exosomes of NC, were not immunosuppressive. Thus, MTEX, present in excess in plasma of patients with metastatic melanoma, mediate immune suppression and contribute to immune escape of the tumor.

Citation Format: Priyanka Sharma, Brenda Diergaarde, Soldano Ferrone, John M. Kirkwood, Theresa L. Whiteside. Effects of melanoma cell-derived exosomes in melanoma patients’ plasma on immune cell functions [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 3091.
Abstract 3098: Maximizing the sensitivity and utility of tissue biopsy by analyzing RNA and incorporating multiple immunotherapy markers
Authors: Kasbek Christopher , Chen Cai , Almalki Nouran , Cortese Jillian , Huang Jun
Published: August 2019

Next-generation sequencing panels are becoming more widely accepted as diagnostics for guiding personalized medicine in oncology. Some weaknesses of current NGS panels include: a lack of prediction of the patient’s response to the recently approved PD-1 checkpoint inhibitors, low sensitivities of fusion variant detection due to the complexity of the translocation events that can occur at the DNA level, and the need for matched normal sample. We are developing a 371-gene panel that uses both DNA and RNA from a single non-matched FFPE sample to guide targeted and immunotherapy decisions. Fusions are identified in RNA, while copy number variation, tumor mutation burden (TMB), microsatellite instability (MSI), HLA typing, single-nucleotide variants, and small insertion/deletion variants are identified in DNA. All exons plus UTR regions of 371 genes are covered at a mean depth of >500X. To validate the TMB test, whole-exome sequencing data from 376 colon adenocarcinoma samples was analyzed with our pipeline using a 4-fold cross-validation design. TMB-high vs. TMB-low was called with 95% accuracy, compared to 90% accuracy of a competitor’s panel. For MSI, the canonical five mononucleotide-repeat sites are interrogated with a limit of detection of 2-5%. RNA samples with Dv200 >10% can be used to identify fusion variants. RNA is converted to cDNA and hybridized with a mixture of probes containing ≈300 well-known fusion junctions and full exon sequences of 37 of the 371 genes which are relevant to fusions. This design leads to improved fusion detection compared to the standard DNA detection, while allowing for the identification of novel fusion junctions. Gender and common SNP’s in DNA and RNA are used to prevent sample misidentification. The accuracy and comprehensiveness of this panel should maximize benefit to the patient.

Note: This abstract was not presented at the meeting.

Citation Format: Christopher Kasbek, Cai Chen, Nouran Almalki, Jillian Cortese, Jun Huang. Maximizing the sensitivity and utility of tissue biopsy by analyzing RNA and incorporating multiple immunotherapy markers [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 3098.
Abstract 3112: Prospective immune phenotyping of neuroblastoma children at diagnosis reveals specific immune defects related to the aggressiveness of the disease
Authors: Susini Sandrine , Rochet Isabelle , Verronèse Estelle , Bardin Christine , Rigal Chantal , Conter Cécile , Marec-Bérard Perrine , Bergeron Christophe , Lardy-Cleaud Audrey , Neymarc Séverine , Metzger Séverine , Stephan Jean-Louis , Plantaz Dominique , Caux Christophe , Ménétrier-Caux Christine , Marabelle Aurelien
Published: August 2019

CONTEXT: Neuroblastoma is the most common extracranial solid tumor in children. Patients with low- and intermediate-risk neuroblastoma (LIRNB) have favorable prognosis and an excellent five-year survival rate of more than 90%. However, in the case of high-risk neuroblastoma (HRNB; ~50% of cases), the prognosis of treatment remains unfavorable and the five-year survival rate remains under 40% despite aggressive multi modal therapy.

QUESTION: We wanted to know if there was a defect in immune surveillance that could explain part of the aggressive phenotype of HRNB.

METHODS: We performed a prospective multi-center study on thirty two new cases of neuroblastoma at all stages. We performed a wide phenotyping of immune cells by flow cytometry in fresh whole blood, and fresh whole bone marrow at diagnosis of children diagnosed for neuroblastoma. Cell counts were performed in order to be able to reason in absolute values (G/L) rather than only percentages. RESULTS: Blood and bone marrow from 20 LIRNB and 12 HRNB were prospectively analyzed. We found that specific subsets of circulating immune cells such as myeloid dendritic cells BDCA-1+, plasmacytoid dendritic cells, Teff/Treg CD4+ T-cells, were decreased in HRNB blood compared to LIRNB groups as opposed to other subsets (e.g iNKT, g9d2 T-cells,⋯). More surprisingly, these differences were not due to defects in cell production as there was no difference found for the same cell populations in the bone marrow between LIRNB and HRNB children.

CONCLUSION: Specific immune cell defects are found in the blood from High risk neuroblastoma children that are not present in the blood of low & intermediate risk neuroblastoma children. These defects are not due to bone marrow production impairment. These results suggest that high risk neuroblastoma disease generates an impairment in the immune homeostasis of children. Gene expression analysis of cytokine/chemokine from these neuroblastoma tumors is underway (NCT01295762).

Citation Format: Sandrine Susini, Isabelle Rochet, Estelle Verronèse, Christine Bardin, Chantal Rigal, Cécile Conter, Perrine Marec-Bérard, Christophe Bergeron, Audrey Lardy-Cleaud, Séverine Neymarc, Séverine Metzger, Jean-Louis Stephan, Dominique Plantaz, Christophe Caux, Christine Ménétrier-Caux, Aurelien Marabelle. Prospective immune phenotyping of neuroblastoma children at diagnosis reveals specific immune defects related to the aggressiveness of the disease [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 3112.