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Deregulation of neuroblastoma-derived myc (N-myc) is a leading cause of malignant brain tumors in children. To target N-myc-driven medulloblastoma, most research has focused on identifying genomic alterations or on the analysis of the medulloblastoma transcriptome. Here, we have broadly characterized the translatome of medulloblastoma and shown that N-myc unexpectedly drives selective translation of transcripts that promote protein homeostasis. Cancer cells are constantly exposed to proteotoxic stress associated with alterations in protein production or folding. It remains poorly understood how cancers cope with proteotoxic stress to promote their growth. Here, our data revealed that N-myc regulates the expression of specific components (∼5%) of the protein folding machinery at the translational level through the major cap binding protein, eukaryotic initiation factor eIF4E. Reducing eIF4E levels in mouse models of medulloblastoma blocked tumorigenesis. Importantly, targeting Hsp70, a protein folding chaperone translationally regulated by N-myc, suppressed tumor growth in mouse and human medulloblastoma xenograft models. These findings reveal a previously hidden molecular program that promotes medulloblastoma formation and identify new therapies that may have impact in the clinic. Significance: Translatome analysis in medulloblastoma shows that N-myc drives selective translation of transcripts that promote protein homeostasis and that represent new therapeutic vulnerabilities.

The BRAF kinase plays a critical role in the MAPK signaling pathway and is mutated in ~8% of all human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), which is found in >70% in these cancers. Despite the clinical success of approved small molecule inhibitors of BRAF V600E (vemurafenib, dabrafenib and encorafenib), this remains an area of unmet medical need because nearly all patients progress, due to either primary or acquired resistance. A bifunctional degradation activating compound (or BiDACTM degrader) may address the liabilities of approved drugs by overcoming, or preventing the emergence of, resistance to BRAF inhibitors. Here we describe CFT1946, an orally bioavailable cereblon-based BiDAC degrader of BRAF V600 mutant proteins, and provide an overview of the medicinal chemistry path leading to its discovery. CFT1946 degrades BRAF V600 mutant proteins, while maintaining exquisite selectivity against the proteome, sparing wild type BRAF (BRAF-WT), ARAF, and CRAF. In A375 cells, CFT1946 potently degraded BRAF V600E and inhibited ERK phosphorylation and cell growth while having no effect in the mutant KRAS, BRAF-WT driven cell line HCT116. In the A375 xenograft model, oral delivery of CFT1946 at 10 mg/kg PO BID resulted in deeper and more durable tumor regression compared to a clinically relevant dose of encorafenib. Further evaluation of CFT1946 in an engineered, clinically relevant BRAFi-resistant A375 cell line (endogenous BRAF V600E + engineered expression of NRAS Q61K) demonstrated that CFT1946 both degraded BRAF V600E and caused a loss of viability in these cells, while treatment with encorafenib had no effect. In xenografts derived from this BRAFi-resistant cell line, oral dosing of CFT1946 as a single agent led to tumor growth inhibition, while treatment with a clinically relevant dose of encorafenib had no effect on tumor growth. Furthermore, dosing CFT1946 in combination with the MEK inhibitor, trametinib, resulted in significant tumor regression, whereas combining encorafenib with the same dose of trametinib had no effect. The medicinal chemistry campaign resulting in CFT1946 focused on the improvement of in vivo pharmacokinetics and rational linker design to achieve high oral bioavailability in a beyond Rule of 5 heterobifunctional degrader. The preclinical data presented herein support the planned Phase 1/2 clinical trial of CFT1946 for the treatment of BRAF-V600 mutant solid tumors. Citation Format: Yanke Liang, Mathew E. Sowa, Katrina L. Jackson, Jeffrey R. Simard, Bridget Kreger, Ping Li, Laura Poling, Joelle Baddour, Andrew Good, Hongwei Huang, Scott Eron, Christopher G. Nasveschuk, Robert Yu, Mark Fitzgerald, Victoria Garza, Morgan W. O’Shea, Gesine Veits, Jeremy Y. Yap, Moses Moustakim, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe S. Patel, Richard Deibler, Kyle S. Cole, David Cocozziello, Fazlur Rahman, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Roy M. Pollock, Stewart L. Fisher. The discovery and characterization of CFT1946: A potent, selective, and orally bioavailable degrader of mutant BRAF for the treatment of BRAF-driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3425.

Brain metastasis (BrM) is the most common form of brain cancer, characterized by neurologic disability and an abysmal prognosis. Unfortunately, our understanding of the biology underlying human BrMs remains rudimentary. Here, we present an integrative analysis of >100,000 malignant and non-malignant cells from 15 human parenchymal BrMs, generated by single-cell transcriptomics, mass cytometry, and complemented with mouse model- and in silico approaches. Specific single-cell interrogation of metastatic tumor cells provides a framework of 8 functional cell programs that coexist or anticorrelate. Collectively, these programs delineate two functional BrM archetypes, that co-exist within each metastatic tumor, one proliferative and the other inflammatory, that are evidently shaped through tumor-immune interactions. Our study provides a foundation to understand the molecular basis of BrM in patients with tumor cell-intrinsic and host environmental traits. For more details please refer to the original study: H. Gonzalez, et al. Cellular architecture of human brain metastases Cell, 185 (2022), pp. 729-745. Citation Format: Hugo Gonzalez, Wenbin Mei, Isabella Robles, Catharina Hagerling, Breanna Allen, Trine Hauge Okholm, Ankitha Nanjaraj, Tamara Verbeek, Sandhya Kalavacherla, Merel van Gogh, Stephen Georgiou, Mariza Daras, Joanna J. Phillips, Matthew H. Spitzer, Jeroen P. Roose, Zena Werb. Cell archetypes in human brain metastases: Connecting programs in tumor cells with the microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr IA022.

TERT promoter mutation (TPM) is found in over 80% of IDH-wildtype glioblastomas (GBMs) and IDH-mutant oligodendrogliomas (ODs). As TPM-mediated reactivation of telomerase is tumor specific, TPM- tumor cells could be selectively targeted to reverse tumor cell immortalization, especially if this mutation is present throughout the tumor. Previous studies on the clonality of TPM present conflicting results and have relied on minimal tumor sampling. In this study, we use a 3D maximal tumor sampling approach to address this critical issue. We investigated TPM clonality in 253 primary and recurrent tumor samples, from 19 IDH-wildtype GBMs and 9 IDH-mutant ODs. An average of nine regions per tumor were intraoperatively biopsied in a manner that maximally represents tumor geography. The 3D location of each biopsy was then recorded and mapped back to the patient’s pre- and post-operative MRI, allowing 3D characterization of each tumor. For initial screening of TPM, PCR and Sanger sequencing were performed on all 253 samples. Deep amplicon sequencing (Amp-seq) was performed on 179 of these samples, including all those in which TPM was not detectable by Sanger. Each Amp-seq reaction included a TPM amplicon and an amplicon with IDH1 mutation for ODs or an amplicon spanning a chromosome 10 SNP to evaluate chromosome 10 loss in GBMs, which are early clonal events for these tumor types. Tumor purity, estimated by the FACETS algorithm applied to whole exome sequencing, was available for all 253 tumor samples and their patient-matched blood cell DNA. TERT expression was measured by RNA-seq (144 samples) and RNA Scope (49 samples). Correlations between TPM and clonal alterations and TPM and TERT expression were analyzed for significance. Of 253 tumor samples, tumor purity could be estimated for 210 (83.0%), and of these, 100% had TPM detected by Sanger and Amp-seq. For samples whose tumor purity could not be estimated (assigned a value of NA by FACETS), TPM was still detected in 36 of 43 (83.7%) samples. Variant allele frequencies (VAFs) of TPM showed high positive correlation with those of clonal alterations in GBMs (R = 0.85, p < 0.0001) and ODs (R = 0.86, p < 0.0001) as well as with tumor purity in both GBMs (R = 0.91, p < 0.0001) and ODs (R = 0.89, p < 0.0001). TPM VAF showed moderate positive correlation with TERT expression in GBMs (R = 0.52, p < 0.0001) and ODs (R = 0.62, p < 0.0001). TPM also showed a nonlinear relationship with TERT expression only in ODs. RNA Scope detected TERT expression in a subset of cells in both GBMs and ODs. To conclude, TPM is a tumor-wide, clonal mutation in primary and recurrent GBMs and ODs, making TPM tumor cells a possible therapeutic target. TPM VAF is moderately correlated with TERT expression, with a nonlinear relationship seen in ODs, suggesting other variables affect TERT expression. RNA Scope detected TERT expression at the single cell level in situ in GBMs and ODs and therefore could serve as a tumor cell-specific marker, although follow up studies are needed. Citation Format: Christina Leann Appin, Abigail K. Suwala, Stephanie Hilz, Radhika Mathur, Ivan V. Smirnov, Chibo Hong, Nicholas O. Stevers, Anny Shai, Albert Wang, Mitchel S. Berger, Susan M. Chang, Joanna J. Phillips, Joseph F. Costello. 3D whole tumor analysis of the TERT promoter mutation in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3797.

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

The BRAF kinase is a critical node in the MAPK signaling pathway and is mutated in approximately 8% of human cancers including melanoma (~60%), thyroid (~60%), and lung adenocarcinoma (~10%). The most common mutation in BRAF is V600E (Class I), occurring in half of malignant melanomas. This mutation hyperactivates ERK and signals as a RAF inhibitor-sensitive monomer. BRAF inhibitors including vemurafenib, dabrafenib and encorafenib have produced impressive responses in V600X patients, however resistance usually emerges within a year, including RAS mutation, BRAFV600E amplification, and BRAFV600E intragenic deletion or splice variants. These inhibitors are also ineffective against non-V600 BRAF mutants (Class II & III). To address some of these limitations we have developed CFT1946, a bifunctional degradation activating compound (BiDAC™) degrader comprising a BRAF kinase domain targeting ligand linked to a cereblon ligand. CFT1946 is capable of degrading BRAF V600E (Class I), G469A (Class II), G466V (Class III) mutations, and the p61-BRAFV600E splice variant while maintaining exquisite selectivity against the proteome including WT BRAF and CRAF. In A375 cells, CFT1946 potently degraded BRAFV600E (Emax = 26%; DC50 = 14nM at 24hr) and, inhibited ERK phosphorylation (IC50 = 11nM at 24hr) and cell growth (GI50 = 94nM at 96hr) while having no effect in the mutant KRAS driven cell line HCT116. In A375 xenografts, oral delivery of CFT1946 resulted in deeper tumor regressions when dosed at 10 mg/kg PO BID and compared favorably to a clinically relevant dose of encorafenib. We further evaluated CFT1946 in an engineered A375-BRAFV600E/NRASQ61K double mutant model of BRAF inhibitor resistance. CFT1946 was able to degrade BRAFV600E in these cells and was much more effective than encorafenib at inhibiting viability in vitro. In this model, in vivo dosing of single agent CFT1946 caused robust tumor growth inhibition and combination with the MEK inhibitor, trametinib, resulted in tumor regressions. The combination of encorafenib and trametinib showed no activity in the same model. Next, we demonstrated that CFT1946 was able to degrade additional BRAF mutant proteins including G469A (Class II), G466V (Class III), and the p61-BRAFV600E splice variant using heterologous expression in HEK293T cells. Additionally, we also showed that CFT1946, but not encorafenib, inhibited proliferation of the BRAFG466V heterozygous lung tumor cell line H1666. Based on its activity in preclinical models, including models of BRAF inhibitor resistance, and its drug-like properties we are progressing CFT1946 as a candidate for clinical development in patients with solid tumors bearing BRAF V600X mutations. Further, given CFT1946’s activity on non-V600 BRAF mutations, we are continuing to explore CFT1946 and related BiDAC degraders as therapeutic options for patients bearing Class II or Class III BRAF mutations. Citation Format: Mathew E. Sowa, Bridget Kreger, Joelle Baddour, Yanke Liang, Jeffrey R. Simard, Laura Poling, Ping Li, Robert Yu, Ashley Hart, Roman V. Agafonov, Grace Sarkissian, Joe Sahil Patel, Richard Deibler, Kyle S. Cole, Scott Eron, David Cocozziello, Fazlur Rahman, Moses Moustakim, Christopher G. Nasveschuk, Katrina L. Jackson, Mark Fitzgerald, Victoria Garza, Morgan O’Shea, Gesine Veits, Jeremy L. Yap, Andrew J. Phillips, Elizabeth Norton, Adam S. Crystal, Stewart L. Fisher, Roy M. Pollock. Preclinical evaluation of CFT1946 as a selective degrader of mutant BRAF for the treatment of BRAF driven cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2158.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Tumor metabolism is reprogrammed to meet the demands of proliferating cancer cells. In particular, cancer cells upregulate synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) in order to allow for rapid membrane turnover. Nonetheless, we show here that, in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas, which produce the oncometabolite 2-hydroxyglutarate (2-HG), PtdCho and PtdE biosynthesis is downregulated and results in lower levels of both phospholipids when compared with wild-type IDH1 cells. 2-HG inhibited collagen-4-prolyl hydroxylase activity, leading to accumulation of misfolded procollagen-IV in the endoplasmic reticulum (ER) of both genetically engineered and patient-derived IDHmut glioma models. The resulting ER stress triggered increased expression of FAM134b, which mediated autophagic degradation of the ER (ER-phagy) and a reduction in the ER area. Because the ER is the site of phospholipid synthesis, ER-phagy led to reduced PtdCho and PtdE biosynthesis. Inhibition of ER-phagy via pharmacological or molecular approaches restored phospholipid biosynthesis in IDHmut glioma cells, triggered apoptotic cell death, inhibited tumor growth, and prolonged the survival of orthotopic IDHmut glioma-bearing mice, pointing to a potential therapeutic opportunity. Glioma patient biopsies also exhibited increased ER-phagy and downregulation of PtdCho and PtdE levels in IDHmut samples compared with wild-type, clinically validating our observations. Collectively, this study provides detailed and clinically relevant insights into the functional link between oncometabolite-driven ER-phagy and phospholipid biosynthesis in IDHmut gliomas. Significance: Downregulation of phospholipid biosynthesis via ER-phagy is essential for proliferation and clonogenicity of mutant IDH1 gliomas, a finding with immediate therapeutic implications. Cancer Res; 78(9); 2290–304. ©2018 AACR.

Cutaneous T-cell lymphoma (CTCL) is a rare, incurable CD4+ T cell malignancy of the skin with a 5-year survival rate of less than 30% in advanced stages. Immune checkpoint inhibitors, such as anti-PD-1 antibodies, have shown dramatic clinical efficacy in multiple advanced cancers, but the majority of cancer patients do not respond to these treatments. The clinical use of immunotherapies will increase considerably in the near future; therefore, predictive biomarkers of response to stratify patients for treatment are needed to limit potentially devastating adverse effects and reduce costs for healthcare systems. A clinical trial of the anti-PD-1 antibody pembrolizumab in CTCL showed that 38% of patients have a durable clinical response. However, standard tests, including comprehensive immunohistochemistry and single-cell quantification of PD-1 expression, have so far failed to identify a predictive biomarker for pembrolizumab response in this cohort. We reasoned that deep profiling of the CTCL tumor microenvironment (TME) using CODEX–a novel technology that allows for highly multiplexed tissue microscopy with >50 simultaneous parameters–could provide insight into the mechanisms of pembrolizumab response and enable prediction. We analyzed the CTCL TME using a tissue microarray of matched biopsies taken before and after pembrolizumab therapy in 7 responders and 7 non-responders. Imaging of 55 markers allowed discriminating malignant CD4+ tumor cells from reactive CD4+ T cells based on nuclear size and differential expression of CD7, CD25 and Ki-67. Unsupervised machine learning followed by supervised curation identified 21 different cell type clusters with spatial information. Integrating these data using advanced computational analysis revealed 10 distinct, conserved cellular neighborhoods (CNs) in the CTCL TME that changed in frequency and distribution during pembrolizumab therapy. In responders, effector-type CNs, including a tumor/dendritic cell CN and a tumor/CD4+ T cell CN, were significantly increased after treatment. In contrast, in non-responders, an immunosuppressive-type CN enriched in regulatory T cells was significantly increased before and after therapy. Importantly, the global frequencies in the tissues of the cell types defining these CNs were not different between patient groups. In addition, RNA sequencing of matched tissue sections revealed higher expression of effector-type cytokines and chemokines in responders. In sum, highly multiplexed analysis of the CTCL TME architecture in combination with RNA sequencing allows discovering novel, predictive spatial biomarkers of immunotherapy response and will pave the way for future studies that functionally address the identified cell types and cellular interactions. Citation Format: Christian M. Schürch, Darci J. Phillips, Belén Rivero Gutierrez, Magdalena Matusiak, Salil S. Bhate, Graham L. Barlow, Steven P. Fling, Nirasha Ramchurren, Robert H. Pierce, Martin A. Cheever, Michael S. Khodadoust, Robert West, Youn H. Kim, Garry P. Nolan. Cellular neighborhoods predict pembrolizumab response in cutaneous T cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6669.

The mTOR signaling is dysregulated prominently in human cancers including glioblastoma, suggesting mTOR as a robust target for therapy. Inhibitors of mTOR have had limited success clinically, however, in part because their mechanism of action is cytostatic rather than cytotoxic. Here, we tested three distinct mTOR kinase inhibitors (TORKi) PP242, KU-0063794, and sapanisertib against glioblastoma cells. All agents similarly decreased proliferation of glioblastoma cells, whereas PP242 uniquely induced apoptosis. Apoptosis induced by PP242 resulted from off-target cooperative inhibition of JAK2 and protein kinase C alpha (PKCα). Induction of apoptosis was also decreased by additional on-target inhibition of mTOR, due to induction of autophagy. As EGFR inhibitors can block PKCα, EGFR inhibitors erlotinib and osimertinib were tested separately in combination with the JAK2 inhibitor AZD1480. Combination therapy induced apoptosis of glioblastoma tumors in both flank and in patient-derived orthotopic xenograft models, providing a preclinical rationale to test analogous combinations in patients. Significance: These findings identify PKCα and JAK2 as targets that drive apoptosis in glioblastoma, potentially representing a clinically translatable approach for glioblastoma.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized 13C-magnetic resonance spectroscopy (13C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-13C-pyruvate to 5-13C-glutamate, relative to cells expressing wild-type IDH1. 13C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-13C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. Cancer Res; 75(15); 2999–3009. ©2015 AACR.

Cholangiocarcinoma (CCA) is a terminal diagnosis, with 5-year survival rates averaging less than 10%. Thus, developing therapeutic options for cholangiocarcinoma patients is essential. FGFR4 is a heavily N-glycosylated receptor tyrosine kinase that signals through Akt, ERK and STAT3 pathways, and whose overexpression in cancer has previously been observed. The activation and signaling through FGFR4 involve the intracellular tyrosine kinase domain that is activated upon ligand binding the extracellular receptor region. Examination of expression in the TCGA database and our studies in CCA cells demonstrated a role for FGFR4 in promoting tumor cell survival and proliferation. Further, we have observed the presence of a proteolytic cleavage product of FGFR4, that is comprised of the intracellular kinase domain, which we call R4-ICD. Our data and previous studies on recombinant protein demonstrated that the intracellular kinase domain in the absence of the transmembrane and ligand-binding domains is constitutively active. HuCCT-1 cells (human CCA cell line lacking endogenous FGFR4 expression) stably transfected to express R4-ICD had increased proliferation and pro-survival phenotypes when compared to the parental cell line. Of 14 frozen CCA human tumor samples, all showed expression of R4-ICD. Compared to normal liver tissue, R4-ICD expression was greater in 65% (9 of 14) of tumor samples than in normal liver. In addition, total FGFR4 expression was lower in normal cholangiocytes than human CCA samples. Full length FGFR4 expression was observed in 12 tumor samples (85%). Of note, full-length FGFR4 expression levels were far weaker than R4-ICD expression in the 10 samples that showed both signals. Because R4-ICD is produced from full length receptor, we interpret the predominance and higher prevalence of R4-ICD to reflect efficient processing to a stable intracellular kinase fragment. We demonstrated at least two glycoforms of FGFR4, terminal and core, in cell lines, and these were also observed in human tumors. We hypothesized that glycosylation may regulate R4-ICD proteolytic production. Live CCA cells in culture were treated with PNGase F to remove extracellular N-linked sugars from cell surface proteins, including FGFR4. Deglycosylation of FGFR4 was confirmed by immunoblotting. Upon deglycosylation, R4-ICD levels rapidly increased. In summary, CCA cells in culture and in tumors were demonstrated to have high levels of R4-ICD, a constitutively active kinase that promoted tumor cell survival and proliferation. The stable cleavage product of FGFR4, R4-ICD, was increased on receptor deglycosylation. Whether aberrant FGFR4 glycosylation contributes to R4-ICD predominance in tumor samples or to tumor aggressiveness remains to be tested. Further understanding of R4-ICD formation will allow us to better target CCA, possibly by reducing proteolytic processing in conjunction with kinase inhibition. Citation Format: Andrew J. Phillips, Ashley M. Mohr, Mary Anne Phillippi, Justin L. Mott, Keith R. Johnson. N terminal glycosylation regulates FGFR4 cleavage in cholangiocarcinoma [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 856.

A considerable challenge in glioblastoma (GBM) therapy is treatment resistance and tumor recurrence, resulting in dismal patient prognosis. GBM aggression and recurrence is often associated with a mesenchymal phenotype, increased expression of glycoproteins and the presence of glioma-initiating “stem-like” cells. Using patient-derived xenograft models, and immune competent syngeneic and transgenic glioma mouse models, we show that aggressive mesenchymal-like GBMs, reminiscent of recurrent GBM, are mechanically stiffer, present with a bulky glycocalyx and demonstrate a stem-like phenotype. We report that the more aggressive mesenchymal GBMs show increased mechanical signaling and contractility, and their tumors are surrounded by a stiffer ECM that maintains and even further enhances integrin mechanosignaling. Since a large proportion of these bulky glycoproteins are also stem markers, upregulation of the glycoproteins and their modulators leads to enhanced GBM stem-ness. This was evident both in our mesenchymal models of GBM as well as multiple patient datasets which compared paired primary and recurrent GBM RNA sequencing and protein data. In addition to stem-ness, we show that the mechanically “enhanced” GBMs also foster a differential immune landscape, infiltrated by type 2 macrophages, believed to be pro-tumorigenic and immune evasive. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignaling, the GBM immune landscape and a bulky glycocalyx, which promotes a mesenchymal, stem-like phenotype and likely recurrence in GBM patients. Thus, therapeutic strategies to target GBM tissue tension could prevent recurrence, reduce mortality and improve patient outcome. Citation Format: Shelly Kaushik, James Matthew Barnes, Russell O. Bainer, Jason K. Sa, Elliot C. Woods, Fuiboon Kai, Jonathon N. Lakins, Joanna J. Phillips, Valerie M. Weaver. A tension-mediated glycocalyx feedback loop promotes glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1900.

Background: Angiogenesis mediated by vascular endothelial growth factor (VEGF) is a hallmark of several cancers. Efficacy of VEGF inhibitors could be increased by improving affinity or tumor penetration, for example by reducing the size of the drug. Recently, the exceptionally stable anti-VEGF single chain variable fragment (scFv) brolucizumab (RTH258, ESBA1008, Novartis) completed phase 3 clinical trials in ophthalmology and was shown to be more efficacious than the larger aflibercept. The same molecule, named DLX1008, is currently in pre-clinical development in gliobastoma and Kaposi sarcoma (KS) by Cell Medica. KS is the most angiogenic cancer known to date. Carcinogenesis is induced by KS-associated herpesvirus (KSHV) and compromised of endothelial cells expressing high levels of VEGFR. KS is unique because angiogenesis (growth of non-tumor endothelial cells) as well as proliferation of the primary tumor cells are dependent on VEGF. No VEGF antagonist has yet been demonstrated to unequivocally improve overall survival in either KS or glioblastoma, which may be due to limited tumor penetration. Methods: Binding affinity to VEGF-A was determined by kinetic exclusion assay (KinExA). For in vivo studies, we used the L1T2 model of KS, whereby KSHV-positive human cells (L1T2/ ATCC® VR-1802™) are implanted subcutaneously onto NGS mice, and the U87 model of glioblastoma, where human glioblastoma cells (U87/ ATCC® HTB-14™) are implanted subcutaneously or intracranially onto SWISS nude mice. Tumor growth was followed by caliper measurements and pathology evaluated by histochemistry or imaging. Results: DLX1008 binds to VEGF-A165 with a KD of 1.05 pM. Bevacizumab binds with a KD of 32.4 pM. Hence, the affinity of DLX1008 to human VEGF is around 30-fold tighter than that of bevacizumab. DLX1008 reduced growth of L1T2 cells in vivo (p ≤ 0.007 by mixed effects model, n = 10) compared to vehicle control at 15 mg/kg once daily 5x/week and (p≤0.001, n = 10) in a second biological repeat. Imaging showed that the tumor died from the inside out in response to DLX1008. DLX1008 reduced the growth of U87 cells subcutaneously implanted in vivo (p ≤ 0.0021 by ANOVA, n = 10) and improved median survival of mice intracranially implanted with U87 cells (p ≤ 0.00026) compared to control scFv at 50 mg/kg twice daily. Staining of intracranial U87 tumors showed decreased CD31-positive blood vessel density as well as decreased p-VEGFR1 immunoreactivity in vascular structures and glioma cells. Conclusions: The high affinity, highly stable scFv DLX1008 shows highly reproducible anti-tumor activity in in vivo preclinical models of KS and glioblastoma. Efficacy was attributable to reduced angiogenesis. Its small size offers advantages in tissue penetration and distribution as compared to bevacizumab. DLX1008 is an excellent antibody to advance into clinical trials. Citation Format: Anthony B. Eason, Sang-Hoon Sin, Emese Szabó, Douglas J. Phillips, Miriam Droste, Abdijapar Shamshiev, Dirk P. Dittmer, Michael Weller. Antitumor activity of DLX1008, a single chain antibody fragment binding to VEGF-A, in in vivo preclinical models of Kaposi sarcoma and glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4.

BRAFV600E mutations are frequent in pediatric gliomas. We previously showed antitumor activity when using BRAF and/or MEK inhibitors against BRAFV600E glioma syngeneic grafts (Grossauer et al., Oncotarget 2016;7:775839). While early clinical trial reports suggest an encouraging response rate to BRAFV600E inhibitors alone (Burger et al., Oncol Rep 2017;38:3291) and in combination with MEK inhibition (Brown et al., CNS Oncol 2017:6:291), resistance has been reported in BRAFV600E mutant melanoma. Moreover, we reported that resistance to BRAFV600E inhibitor monotherapy occurs in mouse models and is driven by stem-like cells (Lerner et al., Cancer Res 2015;75:5355). Rapid reactivation of MEK pathway and prosurvival signaling via EGFR have been implicated as mechanisms for acquired resistance (Yao et al., Oncotarget 2017;8:583). It is just unclear why stem-like cells survive treatment better than non-stem like tumor cells and how they respond to additional therapies, such as MEK inhibition but also immune checkpoint inhibition. Immunocompetent, syngeneic FVB/N mice were intracranial-engrafted with luciferase-modified BRAFV600E mutant CDKN2A-deficient murine glioma cell line 2341luc and treated with BRAFV600E and/or MEK inhibitors, either alone or in combination, and either with or without PD-L1 (Clone 10F.9G2) and CTLA-4 (Clone 9D9) mAbs. While the combination of BRAF and MEK inhibitors significantly increased the median survival from 46 to 70 days (P Citation Format: Stefan Grossauer, Katharina Koeck, Malek Chouchane, Joanna J. Phillips, Claudia K. Petritsch, Theodore Nicolaides. MAPK pathway blockade effects on glioma stem cells and immunotherapy in BRAFV600E mutant gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 143.

While genomic profiling and therapeutic selection support individualized GBM treatment, such therapeutic decision-making is usually made with reference to tumor obtained from the enhancing core region. GBM is known to be heterogeneous and exhibits a high resistance to standard therapies. To address whether non-enhancing tumor (representing the majority of tumor left behind after surgery) shows distinct genomic characteristics and therapeutic targets compared to the enhancing tumor core, we performed genome-wide exome-sequencing and RNA-sequencing for 12 patients with matched enhancing region and at least one non-enhancing region. Non-enhancing biopsies show a surprisingly high level of tumor content, with a median of 28% tumor cells and 6 of the 22 samples having greater than 50% tumor cells. Cognate non-enhancing and enhancing specimens demonstrated overall concordance in therapeutically actionable alterations (single nucleotide variants) and copy number alterations. However, non-enhancing regions were not genetically identical and did reveal additional and distinct variants compared to enhancing cores. For example, the non-enhancing region of patient 1 showed two nonsense NF1 mutations (R1534X; R2517X) while the enhancing region showed an NF1 frameshift mutation (F1247fs). Clonality analysis by LumosVar also indicated that 7 out of 12 patients harbored dissimilar cellular prevalence patterns between enhancing and non-enhancing regions. In addition, comparison of alternative polyadenylation between enhancing and non-enhancing regions uncovered distinct 3' UTR usage: e.g. SGMS2 and TOB1 tended to have longer 3' UTR in enhancing regions whereas longer 3' UTR of SYNPO and NOS1AP were prevalent in non-enhancing regions. We posit that the enhancing component of glioblastoma probably underrepresents the genomic alterations in patients' tumors. Given non-enhancing tumor is left behind after surgical debulking, genomic profiling of this region would potentially reveal more accurate tumor vulnerabilities and lead to more effective therapy. Supported by a grant from the Ben & Catherine Ivy Foundation. Citation Format: Sen Peng, Rebecca Halperin, Harshil Dhruv, Sara Byron, Christophe Legendre, Joanna Phillips, Michael Prados, Michael Berens, Nhan Tran. Probing the non-enhancing component of glioblastoma: Targeting what is left behind [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 289.

Introduction: Cancer cells up-regulate phospholipid biosynthesis in order to meet the demands of uncontrolled cell proliferation. Unusually, mutant isocitrate dehydrogenase 1 (IDHmut) gliomas down-regulate levels of the phospholipid precursors phosphocholine and phosphoethanolamine relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was therefore to investigate whether the phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) are also down-regulated in IDHmut gliomas. Methods: Immortalized normal human astrocytes and U87 cells expressing IDHwt or IDHmut enzyme were studied. Steady-state phospholipid levels were measured by 31P-magnetic resonance spectroscopy (MRS) of cell extracts. De novo phospholipid biosynthesis was quantified by 13C-MRS of cells labeled with 56μM [1,2-13C]-choline and [1,2-13C]-ethanolamine. ER area was measured by STORM imaging of calnexin fluorescence. Results: Steady-state PtdCho and PtdE levels and de novo phospholipid biosynthesis were significantly reduced in IDHmut cells relative to IDHwt. Concomitantly, the activities of CTP:PC cytidylyltransferase (CCT) and CTP:PE cytidylyltransferase (ECT), the rate-limiting enzymes for phospholipid biosynthesis, were significantly reduced in IDHmut cells. CCT and ECT are localized to the endoplasmic reticulum (ER), the site of phospholipid biosynthesis. We therefore measured ER area by super-resolution STORM imaging and found a significant reduction in IDHmut cells relative to IDHwt while cell size remained unchanged. Autophagy of the ER (ER-phagy) can regulate ER area and we found that IDHmut cells showed increased expression of FAM134b, the ER-phagy receptor and higher autophagic flux. These results suggest that IDHmut cells undergo ER-phagy, leading to reduced phospholipid biosynthesis. Silencing FAM134b or the autophagy proteins Atg5 and Atg7 restored ER area and phospholipid biosynthesis and abrogated clonogenicity of IDHmut cells, linking ER-phagy to reduced phospholipid biosynthesis and pointing to a therapeutic opportunity. In the absence of specific ER-phagy inhibitors, we used the late-stage autophagy inhibitor chloroquine (CQ) to test proof-of-principle and found that CQ significantly inhibited orthotopic IDHmut glioma growth in vivo. Importantly, we observed ER-phagy and down-regulation of phospholipid levels in IDHmut glioma patient biopsies, highlighting the translational validity of our findings. Conclusions: We find that down-regulation of phospholipid biosynthesis is a unique metabolic outcome of ER-phagy in IDHmut gliomas. While autophagy has been linked to glucose and glutamine metabolism, this study is the first to link autophagy to phospholipid biosynthesis. Importantly, our study identifies a rationale for the development of novel therapies targeting ER-phagy in IDHmut gliomas. Citation Format: Pavithra Viswanath, Russell O. Pieper, Joanna J. Phillips, Sabrina M. Ronen. Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1431.

Glioblastoma multiforme (GBM), the most common malignant primary brain tumor, represents a significant disease burden. GBM tumor cells disperse extensively throughout the brain parenchyma, and the need for tumor-specific drug targets and pharmacologic agents to inhibit cell migration and dispersal is great. The receptor protein tyrosine phosphatase μ (PTPμ) is a homophilic cell adhesion molecule. The full-length form of PTPμ is down-regulated in human glioblastoma. In this article, overexpression of full-length PTPμ is shown to suppress migration and survival of glioblastoma cells. Additionally, proteolytic cleavage is shown to be the mechanism of PTPμ down-regulation in glioblastoma cells. Proteolysis of PTPμ generates a series of proteolytic fragments, including a soluble catalytic intracellular domain fragment that translocates to the nucleus. Only proteolyzed PTPμ fragments are detected in human glioblastomas. Short hairpin RNA–mediated down-regulation of PTPμ fragments decreases glioblastoma cell migration and survival. A peptide inhibitor of PTPμ function blocks fragment-induced glioblastoma cell migration, which may prove to be of therapeutic value in GBM treatment. These data suggest that loss of cell surface PTPμ by proteolysis generates catalytically active PTPμ fragments that contribute to migration and survival of glioblastoma cells. [Cancer Res 2009;69(17):6960–8]

The treatment of glioblastoma (GBM) remains challenging in part due to the presence of stem-like tumor-propagating cells that are resistant to standard therapies consisting of radiation and temozolomide. Among the novel and targeted agents under evaluation for the treatment of GBM are BRAF/MAPK inhibitors, but their effects on tumor-propagating cells are unclear. Here, we characterized the behaviors of CD133+ tumor-propagating cells isolated from primary GBM cell lines. We show that CD133+ cells exhibited decreased sensitivity to the antiproliferative effects of BRAF/MAPK inhibition compared to CD133− cells. Furthermore, CD133+ cells exhibited an extended G2–M phase and increased polarized asymmetric cell divisions. At the molecular level, we observed that polo-like kinase (PLK) 1 activity was elevated in CD133+ cells, prompting our investigation of BRAF/PLK1 combination treatment effects in an orthotopic GBM xenograft model. Combined inhibition of BRAF and PLK1 resulted in significantly greater antiproliferative and proapoptotic effects beyond those achieved by monotherapy (P < 0.05). We propose that PLK1 activity controls a polarity checkpoint and compensates for BRAF/MAPK inhibition in CD133+ cells, suggesting the need for concurrent PLK1 inhibition to improve antitumor activity against a therapy-resistant cell compartment. Cancer Res; 75(24); 5355–66. ©2015 AACR.

Glioblastoma Multiforme (GBM, WHO Grade IV glioma) is one of the deadliest adult CNS tumors with a median survival of less than two years. It is largely resistant to aggressive therapeutic regimes, which combine radiation, chemotherapy and surgery. Thus, it is imperative to identify the molecular players and pathways that facilitate GBM resistance and recurrence. Nuclear co-repressor protein NCoR2 is upregulated in GBM and its increased expression correlates with poor prognosis in GBM patients (Alrfaei. Plos one. 2013). NCoR2, through its binding to the histone deacetylase protein HDAC3, mediates transcriptional repression (Zhu et al. Brain Res. 2013). NCoR2 is also involved in maintenance of neural stem cells (Jepson et al. Nature. 2007), which contribute to treatment resistance. Our study employs two distinct lineages of glioma - the less aggressive proneural and the highly aggressive mesenchymal subtypes. We show that NCoR2 is differentially expressed across these subtypes, suggesting a role for NCoR2-mediated transcriptional reprogramming in GBM progression. Moreover, knockdown of NCoR2 leads to phenotypic changes both in vitro and in vivo - including effects on neurosphere formation and proliferation, differential expression of stem-like markers as well as regulation of tumor growth and progression in mice. We also demonstrate that NCoR2 expression is increased upon treatment with temozolomide (used for GBM therapy), indicating a protective pro-tumor role for NCoR2. Finally, we explore the contribution of the GBM microenvironment to GBM progression by varying the three-dimensional properties as well as the rigidity of the extracellular matrix and investigating changes in NCoR2 expression and its regulation of tumor progression. We conclude that NCoR2 regulates GBM progression and resistance to therapy potentially via inducing a stem-like phenotype as well as responding to the tumor-promoting mechanical cues within the GBM microenvironment. Citation Format: Shelly Kaushik, Joanna J. Phillips, Valerie M. Weaver. NCoR2 regulates glioblastoma progression and treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3985. doi:10.1158/1538-7445.AM2017-3985

Purposes of the study: Head and neck squamous cell carcinoma (HNSCC) is the sixth most widespread cancer and leading cause of cancer related death world widely. Regardless of the advancement in treatment processes the overall survival rate of patients having this cancer has not substantially improved in the past few decades. Development of tumor and its progression exclusively resides in a small population of cancer cells, termed as cancer stem cells (CSCs). This population of cells can self-renew and re-form the ordered organization of tumors. CSCs contribute to HNSCC and may offer prominent therapeutic strategy for the management of HNSCC. We previously observed that c-Fos was highly up-regulated in the HNSCC sphere forming cells. In the present study, we explored the role of c-Fos in the tumor initiation in relation with cancer stemness and EMT. Experimental procedures: We examined whether overexpression of c-Fos has an effect on tumorigenicity and stemness. For this, c-Fos was overexpressed in non-tumorigenic HNSCC cell lines and tumor growth was investigated. We also examined the expression of different CSC marker genes expression by Western blot analysis. Summary: Our results demonstrated that exogenous expression of c-Fos in non-tumorigenic MDA1386Tu cells makes the cells tumorigenic in nude mice. Mechanistic investigations showed that c-Fos overexpression in MDA1386Tu cells enhanced epithelial-mesenchymal transition (EMT)/CSC properties and increased numbers of oralspheres when grown in ultra-low binding plate. Conclusions: Collectively, our data strongly demonstrate that c-Fos plays a critical role in the modulation of EMT and cancer stem cell reprogramming in HNSCC cells and enhance tumor growth. Note: This abstract was not presented at the meeting. Citation Format: Naoshad Muhammad, Sourav Bhattacharya, Robert Steele, Nancy Phillips, Ratna B. Ray. C-Fos drives non-tumorigenic head and neck squamous cell carcinoma cells to tumorigenic by promoting cancer stem cells like properties [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5761. doi:10.1158/1538-7445.AM2017-5761

Background: Chordoma is a rare cancer (0.08 per 100k/yr) that originates from the notochord and develops in the skull and spine. Treatment options for chordoma include resection and local radiation therapy; however, recurrence rates following treatment are high and the majority of patients ultimately succumb to the disease. Currently there are no approved chemotherapy options for chordoma, and effective treatment options for patients with recurrent or advanced disease are limited. Recent molecular analyses of chordoma have revealed multiple tractable therapeutic targets, providing rationale for new systemic therapies. However, lack of relevant, validated chordoma models has limited preclinical evaluation. To address this need, the Chordoma Foundation and START have collaborated to establish, bank and characterize chordoma tumor models derived from patient samples and established cell lines. Methods: Chordoma patients undergoing resection or biopsy procedures are identified and consented by the Chordoma Foundation under an IRB-approved protocol; tissue is shipped to START and implanted into immune-deficient mice for patient-derived xenograft (PDX) model establishment and banking. Similarly, models from immortalized chordoma cell lines have been established and banked, along with established PDX chordoma models from START or collaborators. Banked PDX models are anonymously linked to available patient clinical information. All chordoma models are subjected to immunohistochemistry (IHC) to confirm histology and presence of brachyury, a protein highly expressed in chordoma tumors. Following validation, models are used at START for drug sensitivity studies and available to investigators for characterization studies. Results: To date, clinical samples from five chordoma patients have been implanted and two PDX models designated CF322 and CF345 have been established. In addition, xenograft models from the UCH-1 and UCH-2 chordoma cell lines have been established at START. PDX models previously developed at START (ST087) and UCSF (SF8894) have also been validated and banked. Drug sensitivity studies have been initiated these two and the U-CH1 models evaluating agents selected and prioritized through a peer review process established by the Chordoma Foundation. Nominations for additional test agents from academic and industry collaborators are reviewed on a rolling basis and evaluation of selected therapies is funded by the Chordoma Foundation. Conclusion: We have established a collaboration to create, bank and characterize a panel of preclinical chordoma tumor models originating from patient samples and established cell lines. To date we have generated three chordoma models available for drug sensitivity studies. This panel of models is intended to serve as a shared resource to the chordoma research community to enable more rapid preclinical evaluation of therapeutic hypotheses. Citation Format: Michael J. Wick, Melissa Rundle, Lizette Gamez, Armando Diaz, Josh Sommer, Patricia Cogswell, Byron Hann, Joanna Phillips, Kyriakos P. Papadopoulos. Establishment and characterization of a panel of cell-based and patient-derived chordoma tumor models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 627.

Prostate cancer is the leading form of cancer in men. Prostate tumors often contain neuroendocrine differentiation, which correlates with androgen-independent progression and poor prognosis. Matrix metalloproteinases (MMP), a family of enzymes that remodel the microenvironment, are associated with tumorigenesis and metastasis. To evaluate MMPs during metastatic prostatic neuroendocrine cancer development, we used transgenic mice expressing SV40 large T antigen in their prostatic neuroendocrine cells, under the control of transcriptional regulatory elements from the mouse cryptdin-2 gene (CR2-TAg). These mice have a stereotypical pattern of tumorigenesis and metastasis. MMP-2, MMP-7, and MMP-9 activities increased concurrently with the transition to invasive metastatic carcinoma, but they were expressed in different prostatic cell types: stromal, luminal epithelium, and macrophages, respectively. CR2-TAg mice treated with AG3340/Prinomastat, an MMP inhibitor that blocks activity of MMP-2, MMP-9, MMP-13, and MMP-14, had reduced tumor burden. CR2-TAg animals were crossed to mice homozygous for null alleles of MMP-2, MMP-7, or MMP-9 genes. At 24 weeks CR2-TAg; MMP-2−/− mice showed reduced tumor burden, prolonged survival, decreased lung metastasis, and decreased blood vessel density, whereas deficiencies in MMP-7 or MMP-9 did not influence tumor growth or survival. Mice deficient for MMP-7 had reduced endothelial area coverage and decreased vessel size, and mice lacking MMP-9 had increased numbers of invasive foci and increased perivascular invasion, as well as decreased tumor blood vessel size. Together, these results suggest distinct contributions by MMPs to the progression of aggressive prostate tumor and to helping tumors cleverly find alternative routes to malignant progression. Cancer Res; 70(6); 2224–34

Purpose. Activation of the PI3K/mTOR pathway in low-grade gliomas (LGG) provides a rationale for treatment with mTOR inhibitors as in preclinical studies activation of PI3K/AKT/mTOR renders cells more susceptible to mTOR inhibition. In a phase II prospective clinical trial of the mTOR inhibitor everolimus for recurrent LGGs, we performed preliminary analyses to identify early imaging and molecular biomarkers of treatment response. Methods. Thirty-six patients (of a planned 60 patients) underwent biopsy at the time of recurrence and were then treated with everolimus. Serial multimodal magnetic resonance imagining was done every 2 months; the imaging included anatomic, dynamic contrast enhanced (DCE), perfusion-weighted, diffusion-weighted, and MR spectroscopic imaging. Tumor molecular analyses included IDH1R132H mutations, 1p19q co-deletions, and phosphoprotein analyses of activation of the PI3K/AKT/mTOR and MAPK pathways by immunohistochemistry, including p-S6, p-PRAS40, p-4EBP1, and p-MAPK. Results. Histologies at original diagnosis were oligodendroglioma (14), astrocyomta (9), oligoastrocytoma (13). At time of recurrence 9/36 patients had transformed to higher-grade disease. Therapy prior to study participation included radiation in 11 and temozolomide in 23. Twelve patients continue on active therapy, 15 have progressed, and the remaining discontinued drug due to toxicity (n=2), poor follow-up (n=1), refusal of further therapy (n=4), or unrelated intercurrent disease (n=2). Fifteen patients had stable disease for over a year, and 4 had stable disease for over 2 years while on treatment, all despite multiple prior recurrences. Six patients have died, 3 of which had high-grade gliomas at recurrence. Analyses of the first 17 accrued patients showed significant changes in parameters estimated from the DCE data in the T2 lesion in subjects who did not progress in the first 6 months; these changes included reductions in the fractional blood volume (median fBV) and vascular permeability (90th percentile Kps) at 4 months (p Conclusions. In patients with recurrent LGGs treated with everolimus, perfusion parameters may represent early markers of treatment response. Apparent “reversal” of association of p-PRAS40 staining with PFS when everolimus is administered suggests that PI3K/AKT/mTOR pathway activation may serve as a predictive marker of response to mTOR inhibition. Accrual continues and overall efficacy results are pending. Citation Format: Daphne Haas-Kogan, Joanna J. Phillips, Annette Molinaro, Michael Wahl, Sabine Mueller, William A. Weiss, Janine M. Lupo, Mitchel S. Berger, Michael D. Prados, Nicholas Butowski, Jennifer Clarke, Arie Perry, Sarah J. Nelson, Susan Chang. Imaging parameters and molecular correlates as predictive markers of treatment response to mTOR inhibition in adults with recurrent low-grade gliomas. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3520. doi:10.1158/1538-7445.AM2013-3520

Multiple chromosome 17 loci may be involved in ovarian carcinogenesis. Fifty-seven sporadic ovarian epithelial tumors were examined for loss of heterozygosity at 15 loci on chromosomes 17p. Eighty % (39 of 49) of informative tumors had allelic loss in 17p13.3 at D17S30, D17S28, or both loci within this region, including 3 of 7 tumors of low malignant potential and 4 of 5 nonmetastatic carcinomas. The smallest region of overlapping deletions extends from D17S28 to D17S30, a distance of 15 kb. Furthermore, several tumors have breakpoints within the region detected by the D17S30 probe. Chromosome 17p13.3 genes with potential tumor suppressor function include HIC-1, DPH2L (N. J. Phillips et al. Isolation of a human diphthamide biosynthesis gene on chromosome 17p13.3, submitted for publication)/OVCA1, PEDF, and CRK. The HIC-1 coding sequence lies i kb centromeric to the D17S28-S17S30 region of deletion (M. Makos Wales et al., Nat. Med., 1:570-577, 1995) but remains a candidate because 5'-regulatory elements may lie within the critical region. Portions of the DPH2L/OVCA1 coding sequence lie within the D17S28-D17S30 interval. Somatic cell hybrid analysis places PEDF in an interval including D17S28, D17S30, and D17S54, whereas CRK is excluded from this interval. Chromosome 17p13.3 loss precedes TP53 and BRCA1 region deletions because the latter changes are see only in high-stage carcinomas. Microsatellite instability plays only a minor role in sporadic ovarian carcinogenesis because only 1 of 57 tumors showed this finding.

To study genetic changes and the evolution of breast cancer, we assayed for loss of heterozygosity (LOH) in 12 sets of synchronous carcinoma in situ (CIS) and invasive cancer, compared to normal control DNA. Microsatellite markers were used, which map to each nonacrocentric autosomal arm. Eight tumor sets demonstrated LOH of the same allele in both concurrent invasive cancer and ductal CIS, for a total of 18 chromosomal loci. Three of nine tumor sets showed LOH on 11p. In two of these sets, LOH was seen on 11p only in the invasive tumor, not the corresponding CIS. One of these tumors also exhibited allelic loss in the invasive tumor for 4 loci, all of which were retained in the noninvasive tumor. For two tumor sets, LOH was mirrored in matched ductal CIS, invasive tumor, and lymph node metastasis. The maintenance of LOH for certain loci throughout the stages of breast cancer suggests clonality of the cancer cells.

In order to determine which tumor suppressor loci are involved in preinvasive breast cancer, we have assayed for loss of heterozygosity (LOH) in ductal carcinoma in situ (DCIS). Areas of DCIS were microdissected from archival paraffin-embedded tissue. DNA was extracted, and LOH was determined by PCR of microsatellite markers that map to 39 autosomal arms. Either uninvolved lymph node or white cell DNA was used as normal control. A total of 61 samples of DCIS were assayed. The average number of informative tumors examined for each marker was 19 (range, 8-48). The median fractional allelic loss was 0.037. The highest percentage of LOH was shown for loci on 8p (18.7%), 13q (18%), 16q (28.6%), 17p (37.5%), and 17q (15.9%). LOH on 18q was found in 10.7% of informative tumors. Fractional allelic loss was associated with LOH on 17p, with high nuclear grade and with the comedo subtype of DCIS. LOH on 17p correlated with LOH on 17q and on 13q. Additional markers were used for 16q and 17p to determine the smallest common region of deletion. These data provide evidence that tumor suppressor loci that map to these regions are involved in the oncogenesis of breast cancer before progression to the invasive phenotype. Our findings provide additional support that multiple loci on 17p and 16q are involved in the development of breast cancer.

Diffuse infiltrating brainstem glioma arises almost exclusively in children and despite advances in imaging, neurosurgery and radiotherapy, the majority of patients die within 2 years after initial diagnosis. Because of the infiltrative nature and anatomic location in an eloquent area of the brain, most brainstem gliomas are treated without a surgical biopsy. The corresponding lack of tissue samples has resulted in a limited understanding of the underlying genetic and biochemical abnormalities associated with these tumors, and is a substantial obstacle for the development of animal model designed specifically for the preclinical testing of specific molecular target of brainstem gliomas. We established a brainstem glioma cells originated from human biopsy sample of a low grade brainstem glioma. The brainstem glioma cells were subsequently modified with hTERT (human telomerase ribonucleoprotein reverse transcriptase) to immortalize the cells and analyzed genetic and biochemical abnomalities of the cells. The established human brainstem glioma cells were tumorgenic in athymic rodents and the developed brainstem tumor was recapitulated the infiltrative growth of diffuse infiltrating brainstem glioma in patients, and can be monitored for tumor growth noninvasively by bioluminescence imaging (BLI). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4307. doi:10.1158/1538-7445.AM2011-4307

Prostate cancer remains the second leading cause of cancer deaths among American men. Earlier diagnosis increases survival rate in patients. However, treatments for advanced disease are limited to hormone ablation techniques and palliative care. Thus, new methods of treatment and prevention are necessary for inhibiting progression of the disease to a hormone refractory state. For centuries, Ayurveda has recommended the use of bitter melon (Momordica charantia) as a functional food to prevent and treat human health related issues. We have recently discovered an anti-proliferative effect of bitter melon extract (BME) in a number of cancer cell lines, including prostate, without affecting normal cell growth. Treatment of prostate cancer cells with bitter melon extract modulated Bax expression and induced poly(ADP-ribose) polymerase cleavage. TRAMP (transgenic adenocarcinoma of mouse prostate) male mice display prostatic intraepithelial neoplasia (PIN) at 10-12 weeks of age. We examined whether oral administration of BME as a dietary compound inhibits or delay the conversion of normal prostate to PIN in these mice. We have observed prevention of PIN in BME-fed mice as compared to untreated control. Further tissues analyses for mechanism of cancer prevention are in progress and the results will be discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4618. doi:10.1158/1538-7445.AM2011-4618

The proto-oncogene MYCN (N-myc in mice) regulates normal brain development, and is often misexpressed in childhood brain tumors, including medulloblastoma. While most medulloblastoma arise independently of Sonic hedgehog (Shh), about ∼25% of are Shh-dependent and have a more favorable prognosis. We recently induced Shh-independent classic and large cell anaplastic medulloblastoma by driving MYCN from the glial Glutamate Transporter 1 promoter in a tet-regulated transgenic mouse model (Swartling et al, 2010). To characterize cell of origin, we used BrdU labeling in premalignant transgenic mice, demonstrating increased proliferation in cerebellar Bergmann glial cells. Bergmann glia showed high levels of glial marker GFAP as well as the transcription factor Sox9. Whereas neural stem cells (NSCs) from normal perinatal cerebellum contained a high proportion of Sox9-positive cells, primary cultures of tumor spheres showed low levels of Sox9, suggesting that MYCN suppresses Sox9 during medulloblastoma progression. To clarify mechanism, we transduced wild-type (WT) or mutationally-stabilized N-myc-T58A into GFAP-positive NSCs from perinatal murine cerebellum. Transplantation of N-myc-WT NSCs was insufficient for tumor formation. N-myc-T58A cerebellar NSCs generated both Shh-dependent and Shh-independent medulloblastoma, as a function of prenatal or postnatal cerebellar age, respectively. Expression of Sox9 correlated directly with Sonic Hedgehog (Shh) dependence in both murine and human medulloblastoma samples. In addition, forced expression of Sox9 induced Shh-dependence and self-renewal in the Shh-negative mouse medulloblastoma spheres. Our data suggest 1). A glial cell of origin for MYCN-driven medulloblastoma, and 2). Sox9 as a critical target for MYCN during medulloblastoma initiation, with activation of Sox9 leading to Shh-dependence, and inhibition of Sox9 leading to Shh-independent medulloblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3451. doi:10.1158/1538-7445.AM2011-3451

BNC105 is a tubulin polymerization inhibitor. This agent selectively disrupts the vasculature in tumors, and in addition acts as a direct cytotoxic against cancer cells. BNC105 has demonstrated single agent efficacy in a number of cell lines and tumor models, including breast, colon, prostate, brain and lung cancer. We have investigated the activity of BNC105 in renal cancer cell lines and renal tumor xenografts. BNC105 exhibits cytotoxic activity with EC50 values in the range of 1-20nM. A number of other chemotherapeutic and targeted agents exhibit activity that is comparable or considerably lower than BNC105. Furthermore, we investigated the activity of BNC105 in disrupting the vasculature of tumors arising from human renal cancer cell lines. Our investigations included the renal cell lines Caki-1 and A498. Caki-1 cells express the wild type of the VHL gene, whereas A498 cells expresses a non-functional mutant form of VHL. In addition A498 cells do not express HIF1alpha. Somatic mutation of the VHL gene is the most frequent genetic alteration observed in renal cell carcinoma leading to inappropriate accumulation of HIF1alpha and HIF2alpha. Single dose BNC105 treatment of mice bearing tumor xenografts arising from these cell lines resulted in substantial tumor vascular disruption giving rise to large areas of tumor necrosis. Immunohistochemistry analysis revealed that expression of HIF1alpha was dramatically increased following BNC105 treatment in tumors arising from the renal cell line Caki-1. HIF1alpha expression in A498 tumors was restricted to endothelial cells and did not change following BNC105 treatment. HIF2alpha expression was found to be upregulated in cells surrounding areas of BNC105 induced necrosis in both Caki-1 and A498 tumors. These findings suggest that vascular disruption and necrosis caused by BNC105 lead to an angiogenic rebound effect that may contribute to tumor recovery from the vascular disruption insult. Given the key role of mTOR signaling in upregulation of HIF we examined the expression of phosphorylated mTOR in renal tumor xenografts following treatment with BNC105. Expression of phosphorylated mTOR was distinctly upregulated in viable cell zones surrounding tumor VDA-induced necrotic areas in both Caki-1 and A498 tumors. These findings suggest that tumor recovery from the necrotic insult of BNC105 is mediated through upregulation of mTOR activity. The potential benefit of combining BNC105 with mTOR inhibitors on the growth of renal tumors is currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2498.

The initial metabolic products of cyclophosphamide (4-hydroxy-cyclophosphamide and aldophosphamide) were prepared biologically in unpurified form. Their toxicity to tumor cells were tested by bioassay techniques and in cell culture, and the deactivation abilities of various tissue-soluble fractions were quantitated. Liver and kidney cytosol effectively deactivated the primary metabolites, whereas cytosols from gastrointestinal tract mucosa, Walker ascites tumor, and spleen were less efficient. When [14C]cyclophosphamide was activated and incubated with liver cytosol, 34% of all radioactivity was identified as carboxyphosphamide, by mass spectrometry of the methyl ester. Measurement of alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (EC 1.2.1.3) activities by reduced nicotinamide adenine dinucleotide production revealed a qualitative correspondence between aldehyde dehydrogenase activity and deactivation ability. Unpurified aldophosphamide and the analogs prepared from 6-methyl- and 5,5-dimethylcyclophosphamides were substrates for nicotinamide adenine dinucleotide-requiring enzymes, whereas incubation of 4-hydroxy-4-methylcyclophosphamide in an unfractionated incubation mixture with liver soluble enzymes did not cause reduced nicotinamide adenine dinucleotide production.

Nine surgically resected Wilms' tumors (WIT) and nude mouse heterotransplants from one WIT were studied by histochemistry and immunohistochemistry. Histochemistry showed acid phosphatase in all cells, while alkaline phosphatase and gamma-glutamyl transpeptidase were present in only some tubules. Using immunohistochemistry, antibodies to the intermediate filaments cytokeratin and vimentin distinguished tubular epithelium and mesenchyme, respectively. WIT tubules were also identified using antibody against a structural component (epithelial membrane antigen) and a secretory product (uromucoid) associated with distal convoluted tubules of normal kidney. Basement membrane surrounding the tubules of WIT was demonstrated using antibody to type IV collagen plus laminin. Different blastema subpopulations were negative or stained positively with antibodies to cytokeratin and vimentin. Production of basement membrane by blastema was also shown. Fetal antigen expression in WIT was examined using the monoclonal PI 153/3 and J5 antibodies. The blastema and tubules of WIT were strongly stained by PI 153/3, which did not label normal adult kidney, and weakly stained by J5, which strongly labeled glomeruli and proximal convoluted tubules of normal kidney. These studies show that WIT blastema is heterogeneous in intermediate filament subtypes, while WIT tubules more closely resemble distal than proximal convoluted tubules of adult kidneys but also retain expression of fetal antigens.

Eighteen Wilms' tumors (WIT), including classical triphasic WIT (blastema, tubules, and mesenchyme) and WIT variants (blastema and tubules, monomorphous tubules, multiloculated cysts, rhabdomyomatous WIT, and clear cell sarcoma), were heterotransplanted in nude mice. Ten (56%) tumors grew and were serially passaged. With two exceptions, the histology of the surgically resected tumors and heterotransplants was found to be similar. Tumors showing a prominent blastema component grew rapidly, whereas those with tubular epithelial or mesenchymal differentiation grew more slowly. Tumors injected s.c. consisted almost entirely of blastema, while tumors injected i.p. consisted of blastema with large areas of tubular epithelium. These results demonstrate that nude mouse heterotransplants of WIT closely resemble the surgically resected tumors from which they derive, that growth rates of WIT heterotransplants depend on the identity of the tumor cells, and that differentiation of WIT heterotransplants can be modulated, depending on the route of administration of tumor cells.

9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-monophosphate (NSC 312887) is a new purine antimetabolite that has been evaluated in a Phase I clinical trial. The schedule of administration consisted of a single i.v. infusion over a period of 30 min once each day for 5 consecutive days, repeated at 4-week intervals. Thirteen patients received 30 courses of the drug in a dose range of 18 to 40 mg/sq m/day. Granulocytopenia and thrombocytopenia were dose limiting. Repeated courses produced similar degrees of granulocytopenia, but in 7 of 7 patients receiving 2 or more courses, the degree of thrombocytopenia was less severe during the first than during subsequent courses. Myelosuppression in humans was more severe than predicted from the mouse model. Lymphopenia was profound at all dose levels, but reversed within 3 weeks. Somnolence occurred during infusion in 8 of 13 patients, but quickly cleared after the infusion was completed. The infused drug was rapidly dephosphorylated in plasma and then cleared so there was no cumulation of drug in plasma when it was rapidly infused once each day in these doses. Phase II studies of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-monophosphate are planned at a starting dose of 18 mg/ sq m/day for patients with prior chemotherapy or radiotherapy and 25 mg/sq m/day for those without prior therapy, as a single dose on each of 5 consecutive days repeated at 21- to 28-day intervals.