bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A81: Identification of reversion mutation by next-generation sequencing in PARP-inhibitor resistant BRCA2 associated breast cancer: Defects in mis-match repair and acquired chemoresistance in breast cancer
Authors: GHEEYA JINESH S. , Tang Jeremy , Ho Joseph , Wong Serena , Toppmeyer Deborah , HIrshfield Kim , Rodriguez Lorna , Ganesan Shridar
Published: May 2016

Women harboring germline mutations in BRCA2 have an increased risk of developing breast and ovarian cancers. The cancers that arise have undergone loss of the wildtype BRCA2 allele, consistent with BRCA2 functioning as a tumor suppressor. The DNA repair defect present in BRCA2-associated cancers renders them highly sensitive to certain classes of DNA damaging agents including platinum agents and PARP inhibitors. BRCA2-mutant cancers have shown acquired resistance to platinum agents through reversion mutations, in which a second mutation in BRCA2 restores an open reading frame that can partially or completely restore functional protein. This kind of functional restoration is often achieved by genomic deletions that excise the exon containing the deleterious germline mutation and place the gene back in frame.

Here we report a case of a BRCA2-mutation associated breast cancer that progressed after a prolonged response to PARP inhibitor- based therapy and became chemoresistant. The patient was positive for germline BRCA2 Q2899 * mutation. Next-generation sequencing of the chemoresistant tumor revealed a secondary mutation in BRCA2 that altered BRCA2 Q2899* to Q2899L_converting a non-sense mutation into a missense mutation. In addition to BRCA2 mutation, the tumor had a deletion in MLH1, suggesting that an acquired defect in mis-match repair may have allowed reversion of BRCA2 Q2899* by a point-mutation in this tumor. Analysis of serial specimens from this patient allows reconstruction of the evolution of this tumor, and points to a novel mechanism of reversion mutation due to defects in mismatch repair. This finding suggest how mismatch repair defects can functionally affect BRCA2 mutant cancers, and raise possible therapeutic strategies.

Citation Format: JINESH S. GHEEYA, Jeremy Tang, Joseph Ho, Serena Wong, Deborah Toppmeyer, Kim HIrshfield, Lorna Rodriguez, Shridar Ganesan. Identification of reversion mutation by next-generation sequencing in PARP-inhibitor resistant BRCA2 associated breast cancer: Defects in mis-match repair and acquired chemoresistance in breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A81.
Abstract A83: Digital quantitative immunohistochemistry for analysis of CCSP-2 and 15-PGDH expression in colon cancer
Authors: Park GyeongMin , Cho Charles Jungje , Shin Ji-Young , Do Eun-Ju , Ryu Yeon-Mi , Lee Gil-Je , Kim Sang-Yeob , Yun Youngkuk , Myung Seung-Jae
Published: May 2016

The analysis using quantitative immunohistochemistry (IHC) with diagnostic and prognostic biomarkers is a powerful tool for scientific and clinical research in colon cancer at early stages. The potential utility of distinct protein expression depending on the stages of colon cancer has generated interest in diagnostic and prognostic biomarkers of colon cancer. Colon cancer secreted protein-2 (CCSP-2) has been reported as a novel protein expressed in colon adenomas and cancers but not in normal colon tissue. Thus, this protein has emerged as a potential prognostic marker at early stages. In addition, 15-hydroxyprostaglandin dehydrogenase (15-PGDH) has been useful as a diagnostic or prognostic marker for colon cancer. To clarify the assessment parameters for CCSP-2 and 15-PGDH in colon cancer, we evaluated its clinical, molecular and survival associations in colon cancer, according to stages of colon cancer and the tumor microenvironment, such as immune cells, by multiplex immunohistochemistry. Formalin fixed paraffin embedded (FFPE) samples of patients' primary tumor were obtained and stained for markers include CD3, CD8, Cytokeratin, Ki-67, CCSP-2 and 15-PGDH by using PerkinElmer Opal system. Digital imaging and analysis were done using PerkinElmer Nuance and Inform software system. Density plot analysis revealed distributions of CCSP-2 and 15-PGDH in colon cancer tissues. The expression of CCSP-2 and 15-PGDH correlated with stages of colon cancer. Thus, CCSP-2 and 15-PGDH, as proteins that distinctly expressed in colon adenomas and adenocarcinomas, may be useful diagnostic and prognostic markers for detection of colorectal tumors.

Citation Format: GyeongMin Park, Charles Jungje Cho, Ji-Young Shin, Eun-Ju Do, Yeon-Mi Ryu, Gil-Je Lee, Sang-Yeob Kim, Youngkuk Yun, Seung-Jae Myung. Digital quantitative immunohistochemistry for analysis of CCSP-2 and 15-PGDH expression in colon cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A83.
Abstract A96: First-in-class orally available immune checkpoint antagonists for cancer therapy
Authors: Sasikumar Pottayil G. , Naremaddepalli Sudarshan S. , Gowda Nagaraj , Adurthi Sreenivas , Dhodheri Samiulla , Dhudashia Amit , Ramachandra Raghuveer , Ramachandra Murali
Published: May 2016

Highly durable clinical responses observed with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have revolutionized the outlook of cancer therapy. However, while these antibodies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection and immune-related adverse events due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months are likely contributing to irAEs observed.

Herein we report the discovery of the first-in-class small molecule PD-L1 antagonists that are amenable for oral dosing to achieve potent anti-tumor activity but with a shorter pharmacokinetic profile as a strategy to better manage irAEs.

A focused library of compounds mimicking the interaction of PD1 with PD-L1 was designed and synthesized. Screening and analysis of the resulting library led to the identification of compounds capable of functional disruption of the PD-L1 signaling. Further optimization of the initial hits resulted in compounds with desirable physico-chemical properties and exposure upon oral administration.

Disruption of the PD1-PDL1 interaction by lead compounds was confirmed in binding assays. Potent activity comparable to that obtained with an anti-PD1 antibody in rescuing lymphocyte proliferation and effector functions inhibited by PD-L1 was observed with lead compounds. In a panel of functional assays, the lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, TIM3, LAG3 and BTLA. In syngeneic pre-clinical models of melanoma and colon cancers, significant efficacy comparable to that observed with an anti-PD1 antibody in inhibition of both primary tumor growth and metastasis was noted upon once a day oral dosing. In a 14-day repeated dose toxicity studies, the lead compound was well tolerated at >100x of the efficacious doses.

The findings demonstrating the inhibition of PD-L1 pathway resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. IND-enabling studies with one of the lead compounds are underway towards advancing it to the clinic.

Citation Format: Pottayil G. Sasikumar, Sudarshan S. Naremaddepalli, Nagaraj Gowda, Sreenivas Adurthi, Samiulla Dhodheri, Amit Dhudashia, Raghuveer Ramachandra, Murali Ramachandra. First-in-class orally available immune checkpoint antagonists for cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A96.
Abstract B100: ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models
Authors: Thompson Scott K. , Jaleel Mahaboobi , Nyavanandi Vijay Kumar , Ramachandra Murali , Subramanya Hosahalli , Basavaraju Aravind , Sihorkar Vaibhav , Smith Roger A. , Rao Niranjan , Gupta Sandeep , Reddy Sanjeeva P.
Published: May 2016

Various genes in RAS-RAF and PI3K pathways are frequently mutated in a wide variety of solid tumors. Concurrent double mutations are also observed quite often in a broad range of tumor types. Combined inhibition of both pathways has been shown to impart greater efficacy in multiple tumor models in animals. In order to simultaneously block these pathways, we have discovered and characterized several molecules showing dual inhibition of the RAS-RAF and PI3K pathways. ASN003 has been identified as a lead compound with potent and highly selective inhibitory activity against B-RAF, PI3K and mTOR kinases (low nM IC50). Within the PI3K family, ASN003 has high selectivity for inhibition of PI3Kα and PI3Kδ over PI3Kβ. In cell-based mechanistic studies, ASN003 did not increase phospho-ERK levels in the KRAS mutant cell line HCT116 and thus may not cause paradoxical activation of RAS/MAPK pathway in tumors. Consistent with its dual inhibition profile, ASN003 showed strong antiproliferative activity in cell lines with B-RAF and PI3K pathway mutations as well as vemurafenib (B-RAF inhibitor)-resistant cell lines. ASN003 suppressed the phosphorylation of downstream targets of B-RAF, PI3K and mTOR in pharmacodynamic studies in multiple tumor models, indicating appropriate target engagement. In in vivo efficacy studies, ASN003 showed regression in a B-RAFV600E mutant A375 xenograft model and also caused significant tumor growth inhibition in RKO and A2058 tumor models, which harbor mutations in PIK3CA or PTEN genes, respectively, in addition to the B-RAF V600E mutation. Dual targeting of the B-RAF and PI3K pathways with ASN003 has the potential to treat and/or prevent the acquired resistance to selective B-RAF inhibitors, and may also treat a broader patient population and provide greater efficacy and survival benefit than selective B-RAF inhibitors or selective PI3K pathway inhibitors alone. Due to the lack of paradoxical activation, treatment with ASN003 may not cause skin toxicities seen with pure B-RAF inhibitors. ASN003 is currently in preclinical development and is expected to enter Phase I/II clinical trials by early 2016.

Citation Format: Scott K. Thompson, Mahaboobi Jaleel, Vijay Kumar Nyavanandi, Murali Ramachandra, Hosahalli Subramanya, Aravind Basavaraju, Vaibhav Sihorkar, Roger A. Smith, Niranjan Rao, Sandeep Gupta, Sanjeeva P. Reddy. ASN003, a unique B-RAF inhibitor with additional selective activity against PI3K and mTOR kinases, shows strong antitumor activity in multiple xenograft models. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B100.
Abstract B11: Deep phenotyping of dissociated cells from PDX model solid tumors and human breast tumors using flow cytometry
Authors: Hahn Friedrich , Snowden Eileen , Porter Warren , Ferguson Mitchell , Dillmore Shannon , Middlebrook Aaron , Niknam Shahryar , Llontop Peter , Ghanekar Smita , Blaesius Rainer
Published: May 2016

The model of solid tumors as monolithic entities under the control of a handful of driver genes which seemed to dominate the general perception of cancer for many years is increasingly being replaced by that of highly diverse ecosystems of cells. Multiple tumor subpopulations, as well as attending non-cancerous cells such as fibroblasts, endothelial cells, and cells from the immune compartment populate this ecosystem, communicate with each other and all influence clinically relevant decision points throughout tumorigenesis. Progress in analyzing and characterizing this highly heterogeneous, complex “society of cells” has been remarkably enhanced using the strengths of flow cytometry. A rather comprehensive characterization of individual cells within a population, known as deep phenotyping, is becoming possible with recent advances in multi-parametric staining, collection, and analysis of solid tissue derived cells. This technology is enabling researchers to identify multiple targets within a single sample more efficiently than with more traditional methods.

We have developed a process whereby single cells are liberated from solid tumors through a combination of mechanical and enzymatic treatments and then interrogated by flow cytometry using deep phenotyping. Our chosen marker panels include targets (CD24, CD44, CD49f, EpCAM, CD166, CD133, CD184, HER2/Neu) which have been used to investigate properties relevant for cancer stem cells (CSC), Endothelial Mesenchymal Transition (EMT) and Tumor Microenvironmental (TME) processes but options are built into the design to accommodate less well characterized targets such as GD2, CD73, Notch receptors, EphB2, and c-Met.

We have identified discrete subpopulations in breast cancer patient-derived xenograft (PDX) tumors in mice, demonstrating consistent and reproducible results which constitute a distinct immunophenotypic fingerprint for every model. Building on the experience with PDX derived biopsies and adding markers targeting the immune system we applied our work flow to clinical breast cancer tissue. Our results demonstrate that the multi-dimensional analyses enabled by a surface marker panel reveals differences in highly characterized subpopulations, some at less than 1% of the total population, that remain hidden by more conventional assessment methods.

Citation Format: Friedrich Hahn, Eileen Snowden, Warren Porter, Mitchell Ferguson, Shannon Dillmore, Aaron Middlebrook, Shahryar Niknam, Peter Llontop, Smita Ghanekar, Rainer Blaesius. Deep phenotyping of dissociated cells from PDX model solid tumors and human breast tumors using flow cytometry. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B11.
Abstract B122: Sumoylation regulates nuclear signaling of ErbB4 intracellular domain
Authors: Knittle Anna M. , Sundvall Maria , Vaparanta Katri , Elenius Klaus
Published: May 2016

The members of the ErbB family (EGFR/ErbB1, ErbB2, ErbB3, and ErbB4) of receptor tyrosine kinases (RTK) regulate key cellular processes including proliferation, differentiation, and survival, and play critical roles in several human malignancies. The alternatively spliced ErbB4 JM-a isoform was the first RTK found to signal by a mechanism involving proteolytic release of an intracellular receptor domain (ICD) upon ligand binding. The released ErbB4 ICD can translocate into the nucleus, where it functions as a transcriptional coregulator. ErbB4 ICD has been shown to regulate mammary epithelial development in vivo. Furthermore, nuclear ErbB4 immunoreactivity is frequently observed in clinical breast cancer samples. Although these findings suggest that ErbB4 signaling via ICD is biologically significant, the factors that regulate subcellular distribution and nuclear functions of ErbB4 ICD are largely unknown.

We hypothesized that ErbB4 ICD is likely to be regulated by different mechanisms than the ones controlling conventional RTK signaling. One regulatory mechanism that is often employed by nuclear proteins is the small ubiquitin-like modifier (SUMO) system. SUMO proteins modify protein substrates covalently, and alter their function through changes in activity, stability, or subcellular localization.

Here, we demonstrate by pull-down and western blotting of cell culture lysates that ErbB4 ICD is modified by SUMO. Full-length ErbB4 was not sumoylated, indicating that SUMO is a specific regulatory modification for the soluble ICD. By using site-directed mutagenesis followed by sumoylation assays, we mapped the major SUMO acceptor site in ErbB4. Wild type and sumoylation deficient mutant of ErbB4 did not differ in their ability to activate canonical RTK signaling pathways downstream of ErbB4. However, fractionation experiments of MCF-7 breast cancer cells showed weaker nuclear targeting of sumoylation deficient mutant compared to wild-type ErbB4 ICD. Protein inhibitor of activated STAT3 (PIAS3), a SUMO E3 ligase that could stimulate the ErbB4 ICD sumoylation, modulated the transcriptional coregulatory activity of ErbB4 ICD in luciferase reporter assays, as well as the function of ErbB4 ICD in HC11 mouse mammary epithelial cell differentiation. Based on these findings we propose that the SUMO system regulates nuclear localization and signaling of ErbB4 ICD.

Citation Format: Anna M. Knittle, Maria Sundvall, Katri Vaparanta, Klaus Elenius. Sumoylation regulates nuclear signaling of ErbB4 intracellular domain. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B122.
Abstract B141: Predictive value of genetically engineered endogenous mouse models in preclinical therapeutic studies
Authors: Gupta Aayush , Trajkovic-Arsic Marija , Heid Irina , Teichman Nicole , Kalederis Evdokia , Braren Rickmer , Siveke Jens
Published: May 2016

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies with an alarming resistance to both chemotherapeutics and targeted therapy approaches. Genetically engineered mouse models (GEMM) of PDAC reflect molecularly and pathophysiologically the carcinogenesis and cancer progression observed in humans, thus being excellent tools for preclinical evaluation of new therapies.

To successfully predict and characterize the effect of chemotherapies and targeted approaches in PDAC, we use a complex and highly aggressive GEMM of endogenous PDAC, Ptf1a +/Cre Kras +/LSL-G12D p53Lox/Lox (CKP) mice. To do so, we established a highly usable GEMM-based therapy platform including multi-parametric MR imaging using CKP mice, in which tumors develop within the first 6 weeks of age (Bardeesy et al, 2006).

Animals receive a T2w scan on a 3T clinical MRI scanner for detection and staging of solid tumors for study enrolment at defined inclusion criteria (tumour volume 200-400 mm3). Mice get a weekly scan throughout the study until endpoint criteria are met. Upon reaching the endpoint criteria, mice are sacrificed and tumor material is processed for histopathological and RNA/Protein analysis, isolation and culturing of primary tumor cells. Tumors are assessed macro- and microscopically and graded. Proliferation, apoptosis and analysis of respective downstream effectors are characterized. Further comprehensive analysis depending on the respective phenotypical features is then carried out. Primary cell lines are generated for further functional and molecular characterization including but not limited to cell viability, drug sensitivity, expression and methylation profiling.

Through this platform, we have successfully been able to target key signaling pathways and to identify promising novel chemotherapeutic and targeted combinations for PDAC. Here we want to highlight the vital need to develop improved preclinical tools to characterize individual tumors throughout the course of treatment. We propose that such a platform is highly relevant and usable for predicting responses to chemotherapeutic and targeted agents.

Citation Format: Aayush Gupta, Marija Trajkovic-Arsic, Irina Heid, Nicole Teichman, Evdokia Kalederis, Rickmer Braren, Jens Siveke. Predictive value of genetically engineered endogenous mouse models in preclinical therapeutic studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B141.
Abstract B156: TP53 loss creates therapeutic vulnerability in colorectal cancer
Authors: Liu Yunhua , Lu Xiongbin
Published: May 2016

TP53, a well-known tumour suppressor gene, is frequently inactivated by mutation or deletion in a majority of human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment due to the complexity of p53 regulators and their poor drugability. Here, we demonstrate that genomic deletion of TP53 frequently encompasses neighbouring essential genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such an essential house-keeping gene that is virtually co-deleted with TP53 in many types of human cancer. It encodes the largest and catalytic subunit of RNA polymerase II complex, which is specifically inhibited by α-Amanitin. Our analysis of clinical samples and cancer cell lines in The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopaedia (CCLE) reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal tumours. Suppression of POLR2A with α-amanitin or short-hairpin RNAs, selectively inhibits proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited due to its liver toxicity. However, we found that α-amanitin-based antibody drug conjugates (ADCs) are highly effective therapeutic agents with dramatically reduced toxicity. Here, we show that low doses of α-amanitin-conjugated anti-EpCAM (Epithelial Cell Adhesion Molecule) antibody lead to complete tumour regression in murine models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a novel therapeutic approach for cancers harbouring such common genomic alterations.

Citation Format: Yunhua Liu, Xiongbin Lu. TP53 loss creates therapeutic vulnerability in colorectal cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B156.
Abstract B16: Cetuximab activates Foxo3a via the MAPK p38 to induce apoptosis and reduce cell proliferation in colorectal cancer cells
Authors: Marzi Laetitia , Combes Eve , VIE Nadia , Tosi Diego , Ayrolles-Torro Adeline , Larbouret Christel , Denis Vincent , Linares Laetitia K. , Montagut Clara , Iglesias Mar , Martineau Pierre , Lam Eric W-F , Del Rio Maguy , Gongora Céline
Published: May 2016

Cetuximab is a monoclonal antibody against EGFR and is associated with 5-FU and irinotecan for the treatment of patients with colorectal cancer harboring wild type KRAS. However, cetuximab is ineffective in patients with constitutively active mutated KRAS and also in half of the patients with wild type KRAS. As cetuximab downstream cellular targets are not fully characterized, the aim of this study was to identify the signaling pathways activated by cetuximab in CRC cells. Our results show that in addition to inhibiting ERK and AKT, cetuximab also activates the mitogen-activated protein kinase (MAPK) p38. We have showed that p38 inhibition reduced cetuximab efficacy on cell growth and cell death. At the molecular level, cetuximab activates the transcription factor FOXO3a and promotes its nuclear translocation via p38-mediated phosphorylation. FOXO3a phosphorylation and activation by p38 led to the up-regulation of its target genes p27 and BIM and the subsequent induction of apoptosis and inhibition of cell proliferation. Finally, FOXO3a silencing reduced cetuximab cytotoxicity, further confirming FOXO3a role in cetuximab effect. Collectively, our results identify FOXO3a as a key mediator of cetuximab mechanism of action in CRC cells KRAS WT and define p38 as its main activator in this context.

Citation Format: Laetitia Marzi, Eve Combes, Nadia VIE, Diego Tosi, Adeline Ayrolles-Torro, Christel Larbouret, Vincent Denis, Laetitia K. Linares, Clara Montagut, Mar Iglesias, Pierre Martineau, Eric W-F Lam, Maguy Del Rio, Céline Gongora. Cetuximab activates Foxo3a via the MAPK p38 to induce apoptosis and reduce cell proliferation in colorectal cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B16.
Abstract B173: Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2
Authors: Jacoby Douglas B. , Zoubeidi Amina , Corey Eva B. , Mostaghel Elahe , Kwegyir-Afful Andrew K. , Ramalingam Senthilmurugan , Njar Vincent
Published: May 2016

Multiple experimental approaches were used to examine whether and by what mechanism galeterone affects prostate cancer growth in cells and tumors expressing androgen receptor (AR) splice variants, such as AR-V7. Galeterone is a selective, multitargeted, small molecule that disrupts androgen signaling at multiple points in the pathway. ARMOR3-SV is a Phase 3, randomized, open-label, multicenter, controlled clinical trial in metastatic castration resistant prostate cancer (mCRPC) patients whose tumors express AR splice variant-7 mRNA (AR-V7). AR-V7 is a truncated, constitutively active splice variant of the AR that lacks the ligand binding domain (LBD) and has been implicated in prostate cancer progression. The expression of AR-V7 occurs in approximately 14-26% of men with mCRPC prior to second-generation anti-androgens or chemotherapy, and AR-V7 expression has been clinically associated with resistance to enzalutamide (Xtandi) or abiraterone (Zytiga). Here we provide the pre-clinical and clinical rationale for the development of galeterone in mCRPC patients with AR-V7.

Our findings show that using multiple pre-clinical approaches, galeterone reduces in vitro prostate cancer cell proliferation, androgen receptor signaling and xenograft tumor growth using cells and tumors that express AR splice variants. Reductions in AR splice variant protein were also observed in cells that also co-express wild-type AR, and prostate cancer cells that lack wild type AR, but transiently express AR splice variant protein. Clinical data further support activity of galeterone in patients with truncated androgen receptors. Galeterone's unique mechanism of AR protein downregulation occurs in both splice variant and wild-type AR, supporting that galeterone-induced AR downregulation is independent of the AR LBD. Because preclinical and clinical data support an opportunity for treatment in this setting, Tokai Pharmaceuticals designed ARMOR3-SV to test galeterone vs. enzalutamide in men with metastatic castration-resistant prostate cancer whose tumors express the AR-V7 splice variant.

Citation Format: Douglas B. Jacoby, Amina Zoubeidi, Eva B. Corey, Elahe Mostaghel, Andrew K. Kwegyir-Afful, Senthilmurugan Ramalingam, Vincent Njar. Galeterone shows anti-tumor activity in multiple pre-clinical models that express androgen receptor splice variants, supporting correlative patient data seen in ARMOR2. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B173.
Abstract B177: RXDX-107, a dodecanol alkyl ester of bendamustine, demonstrates improved pharmacokinetic properties and significant anti-tumor efficacy in preclinical models of solid tumors
Authors: Martin Leenus , Patel Roopal , Drager Anthony , Roemmele Renee , McIntyre Bradley , McKean Robert , Patel Piyush , Chua Peter , Cao Jerry , Shoemaker Robert , Brown Peter , Multani Pratik , Wild Robert , Lin Ralph , Li Gang
Published: May 2016

Bendamustine is an alkylating agent that can form interstrand DNA crosslinks (ICLs) and cause cell death via several pathways, including intrinsic apoptosis. Bendamustine has proven to be a potent and effective treatment for patients with chronic lymphocytic leukemia (CLL) and rituximab-refractory indolent non-Hodgkin's lymphoma (NHL), with additional clinical activity seen in other B-cell malignancies and in multiple myeloma (MM). However, the activity of bendamustine in the treatment of solid tumor malignancies has been less impressive, possibly due to the pharmacokinetic and biodistribution properties of bendamustine.

RXDX-107 is a dodecanol alkyl ester of bendamustine, and the clinical drug product is encapsulated in human serum albumin (HSA) to form nanoparticles. RXDX-107 was designed to improve tissue biodistribution over bendamustine, which may result in superior efficacy and tolerability in patients with solid tumors. In in vitro anti-proliferative studies, RXDX-107 displayed dose-dependent cytotoxicity against multiple solid tumor cell lines, including non-small lung cancer (NSCLC), breast and ovarian cancer. While the IC50s of RXDX-107 were comparable to those of bendamustine, RXDX-107 displayed stronger induction of pH2AX (a biomarker for DNA damage) and more complete cell killing than bendamustine in multiple cell lines. Furthermore, RXDX-107 potently inhibited subcutaneous tumor growth in cell line-derived xenograft (CDX) models of human NSCLC and breast cancer. Importantly, RXDX-107 also showed single agent anti-tumor activities, including tumor regression in multiple patient-derived xenograft (PDX) models of advanced solid tumors, such as breast, lung, and ovarian cancer.

In conclusion, our preclinical data demonstrate potent and broad anti-tumor activity of RXDX-107 across a variety of solid tumor types, and support further clinical development of this novel drug candidate for the treatment of solid tumors.

Citation Format: Leenus Martin, Roopal Patel, Anthony Drager, Renee Roemmele, Bradley McIntyre, Robert McKean, Piyush Patel, Peter Chua, Jerry Cao, Robert Shoemaker, Peter Brown, Pratik Multani, Robert Wild, Ralph Lin, Gang Li. RXDX-107, a dodecanol alkyl ester of bendamustine, demonstrates improved pharmacokinetic properties and significant anti-tumor efficacy in preclinical models of solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B177.
Abstract B195: Serum levels of CXCL10 and CXCL11 elevate after skin rash induction, the commonest adverse event by bendamustine in the treatment of malignant lymphoma
Authors: Terui Yasuhito , Mishima Yuji , Hatake Kiyohiko
Published: May 2016

Introduction: Bendamustine has been recently approved for the treatment of low-grade malignant lymphoma. In spite of the better efficacy, skin rash is the commonest adverse event by bendamustine in the treatment of malignant lymphoma. To understand the mechanism of the skin rash, we prospectively examine the relationship between skin rash and cytokines/chemokines.

Methods: After informed consent to the patients, who would received bendamustine (B) or bendmustine+rituximab (BR), the sera were collected before and after the treatment of B or BR. The Cytokine/Chemokine array was performed by Bio-Plex system.

Results: 20 patients with FL (n = 18), MCL (n = 1), and low-grade B-cell lymphoma (n = 1) were enrolled in this study. All patients wee treated with B or BR regimen. Nine patients showed skin rash after the treatment of bendamustine. In Cytokine/Chemokine array, serum levels of CCL22 and CCL25 before the treatment were significantly higher in skin rash group (p = 0.010738476 and p = 0.030366028, respectively), and serum levels of CXCL10 and CXCL11 after skin rash were higher significantly (p = 0.000923527 and p = 0.014890795, respectively).

Conclusion: The commonest adverse event by bendamustine and CXCL10 and CXCL11might contribute to induction of skin rash by bendamustine, and inhibition of their receptor, CXCR3 may be helpful for the treatment of skin rash by bendamustine

Citation Format: Yasuhito Terui, Yuji Mishima, Kiyohiko Hatake. Serum levels of CXCL10 and CXCL11 elevate after skin rash induction, the commonest adverse event by bendamustine in the treatment of malignant lymphoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B195.
Abstract B22: Outcomes and genomic mutational profiling results in patients with cancers of the head and neck treated with dovitinib in the Signature Program
Authors: Piha-Paul Sarina A. , Knost James A. , Raez Luis E. , Cohn Allen L. , Tawbi Hussein A. , Parasuraman Sudha , Kang Barinder P. , Matys Renata M. , Slosberg Eric D.
Published: May 2016

In the Signature Program, patients with advanced solid and hematologic cancers without other standard treatment options are enrolled in 1 of 8 tissue-agnostic, mutation-specific protocols. With no predetermined study sites, the program is able to bring the protocol to the patient through local enrollment via physician-directed genomic profiling of tumors. The primary endpoint of each study is clinical benefit, including complete response, partial response, or stable disease [SD], at 16 weeks by investigator assessment using Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 or appropriate hematologic criteria. This novel design allows rapid matching of patients to a relevant targeted therapy. Dovitinib, a tyrosine kinase inhibitor with activity against multiple receptor tyrosine kinases, is currently in clinical development. Eighty patients were accrued in the dovitinib (TKI258) protocol from May 2013 to January 2015. Here, we describe a head and neck cancer subset of this population, including adenoid cystic carcinoma (ADC; n = 6), head and neck squamous cell carcinoma (HNSCC; n = 5), and salivary gland cancer (n = 1). The median age was 62 years, and patients had a median of 2 prior lines of therapy; 69% of patients were male, 85% were Caucasian, and 31% and 69% had an Eastern Cooperative Oncology Group performance status of 0 and 1, respectively. Patients met mutational inclusion criteria, with mutations in PDGFRα/β, c-KIT, VEGFR2, FGFR1/2, and RET. There were no confirmed responses; however, 5 patients had SD on study. Of the 5 patients with SD, 4 had clinical benefit at 16 weeks (1 patient with SD at 8 weeks discontinued before 16 weeks). Consistent with other studies of dovitinib monotherapy, gastrointestinal adverse events (fatigue [69%], nausea [69%], diarrhea [54%], and vomiting [54%]) were the most common. In addition, pretreatment tissue biopsies from all patients were analyzed by a large multigene next-generation sequencing assay. Results to date indicate that the ADC cohort has a lower mutational load and fewer prior therapies than the HNSCC cohort. Further analysis of a correlation between clinical benefit and specific mutational profile is ongoing.

Citation Format: Sarina A. Piha-Paul, James A. Knost, Luis E. Raez, Allen L. Cohn, Hussein A. Tawbi, Sudha Parasuraman, Barinder P. Kang, Renata M. Matys, Eric D. Slosberg. Outcomes and genomic mutational profiling results in patients with cancers of the head and neck treated with dovitinib in the Signature Program. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B22.
Abstract B49: Inhibition of β-catenin pathway to overcome endocrine resistance in tamoxifen-resistant breast cancer cell line
Authors: Won Hye Sung , Lee Kyoung Eun , Nam Eun Mi , Mun Yeung-Chul , Seong Chu-Myong , Lee Soon Nam
Published: May 2016

Background: It is well known that Wnt/β-catenin signaling pathway is involved in hormone receptor negative breast cancer, especially triple-negative breast cancer. In the case of hormone resistant breast cancer cells, they rarely showed hormone receptor expression. So we aimed to investigate whether the β-catenin pathway becomes activated in endocrine-resistant breast cancer cells and inhibition of β-catenin pathway can overcome endocrine resistance.

Methods: We have established an MCF-7-derived tamoxifen-resistant cell line (TamR) by long-term culture of MCF-7 cells with gradually increasing 4-hydroxytamoxifen concentration till 3μM. The levels of protein expression and mRNA transcripts were determined using western blot analysis and real-time quantitative PCR. The transcriptional activity of β-catenin was measured using luciferase activity assay. We used ICG-001 as inhibitor of β-catenin transcription activity.

Results: The expression of estrogen receptor was significantly decreased in TamR cells. On the other hand, the expressions of HER2 and EGFR were increased in TamR cells than in control cells. The active (uncomplexed form) β-catenin level was increased in TamR cells, and also showed a significantly increased β-catenin transcriptional activity. The ICG-001, small-molecular inhibitor of Wnt/β-catenin pathway, treatment significantly reduced β-catenin transcriptional activity in TamR cells. The ICG-001 reduced cell viability fo TamR cells which showed resistance to tamoxifen by 65.3%, and also inhibited target gene cyclin D1 expression. The combination of ICG-001 and mTOR inhibitor, rapamycin reduced cell viability of TamR cells by 81.7% and there was an additive effect of two drugs as a combination index of 1.022.

Conclusions: The β-catenin pathway is activated in endocrine-resistant breast cancer cells and inhibition of that pathway would be a new therapeutic strategy which overcomes endocrine resistance in breast cancer.

Citation Format: Hye Sung Won, Kyoung Eun Lee, Eun Mi Nam, Yeung-Chul Mun, Chu-Myong Seong, Soon Nam Lee. Inhibition of β-catenin pathway to overcome endocrine resistance in tamoxifen-resistant breast cancer cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B49.
Abstract B53: Dynamic evolution of resistance to EGFR blockade from drug tolerant cancer cells
Authors: Hata Aaron N. , Niederst Matthew , Gomez-Caraballo Maria , Archibald Hannah , Siddiqui Faria , Mulvey Hillary , Hu Haichuan , Sequist Lecia , Engelman Jeffrey
Published: May 2016

Despite the success of targeted cancer therapies, acquired drug resistance remains a significant clinical challenge, as in the case of EGFR mutant non-small cell lung cancers (NSCLC) treated with EGFR inhibitor therapy. Although molecular mechanisms of acquired resistance to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. In some cases, clones with clinically validated genetic resistance mechanisms may exist prior to drug exposure and are simply selected by treatment. Alternatively, it has been hypothesized that drug tolerant (or “persister”) cells without bona fide resistance mechanisms may survive initial drug treatment by epigenetic adaptations, and undergo further evolution over time to acquire validated genetic resistance mechanisms. Although this would have immediate implications for new therapeutic strategies to prevent resistance, there has not been any direct evidence that drug tolerant cells can undergo such evolution. Using EGFR mutant NSCLC models, we observe that acquired resistance caused by the T790M gatekeeper mutation, which is observed in 50% of patients with acquired resistance to EGFR inhibitor therapy, can occur by either selection of pre-existing T790M clones or via evolution of T790M-negative drug tolerant cells that develop the mutation during months of drug treatment. Additionally, the path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells have a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR and can be sensitized by BCL-XL inhibitors. In total, these findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to new therapeutic opportunities to prevent or overcome resistance in the clinic.

Citation Format: Aaron N. Hata, Matthew Niederst, Maria Gomez-Caraballo, Hannah Archibald, Faria Siddiqui, Hillary Mulvey, Haichuan Hu, Lecia Sequist, Jeffrey Engelman. Dynamic evolution of resistance to EGFR blockade from drug tolerant cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B53.
Abstract B58: BIM, a key player in apoptosis induction and acquired venetoclax resistance in follicular lymphoma cells
Authors: Bodo Juraj , Zhao Xiaoxian , Souers Andrew J. , Phillips Darren C. , Smith Mitchell R. , Hsi Eric D.
Published: May 2016

The chromosomal translocation t(14;18) in follicular lymphoma (FL) cells is a primary oncogenic event resulting in BCL-2 over-expression. BCL-2 is not directly responsible for growth of tumor cells, but it protects them against death by binding pro-apoptotic proteins. This study investigates in vitro and in vivo activity of the BH3 mimetic venetoclax (ABT-199) and mechanisms of acquiring resistance in FL.

Primary FL cells with similar expression of anti-apoptotic proteins, such as BCL-2 and MCL-1, showed variable sensitivity to treatment, which correlated with expression of BIM. To further study this correlation we used two t(14;18)+ cell lines, WSU-FSCCL and FC-TxFL2. As with primary cells, FC-TxFL2 cells with higher expression of BIM protein were more sensitive to venetoclax-induced apoptosis. The treatment caused dissociation of BCL-2/ BIM complexes, decreased mitochondrial potential, activation of caspase-3 and caspase-7, and PARP cleavage. Interestingly the population of cells that survived venetoclax treatment showed an increase of phospho-ERK1/2 and phospho-BIM (S69), as well as a decrease of total BIM levels.

Venetoclax treatment of murine xenografts of FC-TxFL2 cells resulted in significant delay in tumor growth. However, viable cells isolated from treated tumors showed a decrease of sensitivity to venetoclax exposure (IC50 increased almost 10 times) in comparison with the cells from the mice treated with vehicle only. These cells contained higher levels of MCL-1 and lower levels of BIM protein compared to control cells. BCL-2 levels remained unchanged.

Venetoclax resistant cells prepared in vitro by continuous treatment with increasing concentrations of venetoclax initially showed elevated levels of p-AKT, p-FOX01/3a, MCL-1, dissociation of BIM/BCL-2/BECLIN1 complex, decrease of p62 level (a sign of an increased autophagy) together with a slight decline of BIM expression. After a stable resistant cell line was established, a significant reduction of BCL-2 levels and almost total absence of BIMEL, BIML and BIMS isoforms was observed.

Detailed knowledge of venetoclax action and the mechanisms for acquired resistance in t(14;18)+ cells may help inform biomarker strategies and dosing schedules in future clinical trials. Inhibition of pathways activated in order to protect the FL cells (e.g. by down-regulating BIM (ERK or AKT pathway) or by autophagy) can significantly augment venetoclax potency and decrease incidence of acquired resistance.

Citation Format: Juraj Bodo, Xiaoxian Zhao, Andrew J. Souers, Darren C. Phillips, Mitchell R. Smith, Eric D. Hsi. BIM, a key player in apoptosis induction and acquired venetoclax resistance in follicular lymphoma cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B58.
Abstract B61: Bcr-Abl1 V304D mutation in CML: friend or foe?
Authors: Pricl Sabrina , Laurini Erik , Fermeglia Maurizio , Storici Paola , Giabbai Barbara , Semrau Marta , De March Matteo , Donato Nicholas J. , Quintas-Cardama Alfonso
Published: May 2016

Background

Our recent work revealed that some chronic myeloid leukemia (CML) patients failing imatinib therapy carry the BCR-ABL1 V304D mutation, and is associated with poor clinical outcomes. BCR-ABL1 V304D variant has rarely been described in clinical specimens, and its location within the BCR-ABL1 Kinase Domain (KD) appears to be in an area of the protein not involved in the direct binding with tyrosine kinase inhibitors (TKIs) such as Imatinib and Ponatinib. The present study integrates computational, structural, and molecular biology techniques to investigate the role of the BCR-ABL1 V304D mutation in kinase activity and TKI resistance.

Materials and methods

In silico experiments are used to probe whether, compared to the wild type (wt), the V304D mutation i) directly affects the kinase domain fold and/or thermodynamic stability and ii) if/how these effects might eventually interfere with BCR-ABL1 binding to the TKIs. Small angle X-ray scattering (SAXS), fluorescence, and circular dichroism spectroscopy are employed for structural characterization of the wt/mutated kinase domain and their interaction with TKIs. Cell proliferation, protein phosphorylation, and kinase activity assays are performed (with/without) the TKIs to assess the effect of this mutation on BCR-ABL1 KD, its enzymatic and transforming activities.

Results

Our preliminary results show that BCR-ABL1V304D does not transform Ba/F3 cells, retains minimal kinase activity, and fails to activate downstream pathways. At the same time, the V304D BCR-ABL1 isoform partially protects cells from cytokine withdrawal and exhibits imatinib resistance. The SAXS-based structural model of the kinase domain mutant in complex with Ponatinib does not reveal major structural perturbations compared to the wild type protein. Molecular simulations on the SH2-linker-SH3-KD construct suggest a possible rationale for the TKI resistance seen in the clinic.

Conclusions

The preliminary results obtained indicate that BCR-ABL1V304D results in kinase inactivation, pan-TKI resistance mediated by lower control of protein autoinhibition via perturbation of the SH3 binding domain and very poor prognosis.

Citation Format: Sabrina Pricl, Erik Laurini, Maurizio Fermeglia, Paola Storici, Barbara Giabbai, Marta Semrau, Matteo De March, Nicholas J. Donato, Alfonso Quintas-Cardama. Bcr-Abl1 V304D mutation in CML: friend or foe? [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B61.