Laura Gamble

Therapeutic targeting of the MYC signal by inhibition of histone chaperone FACT in neuroblastoma

Histone chaperone FACT acts in a positive feedback loop with MYCN and is a therapeutic target in neuroblastoma. Uncovering the FACTs in neuroblastoma Neuroblastoma is a common pediatric cancer of the nervous system. It is often difficult to treat, and tumors with amplifications of the MYC oncogene are particularly aggressive. Carter et al. have identified a histone chaperone called FACT as a mediator of MYC signaling in neuroblastoma and demonstrated its role in a feedback loop that allows tumor cells to maintain a high expression of both MYC and FACT. The authors then used curaxins, which are drugs that inhibit FACT, to break the vicious cycle. They demonstrated that curaxins work in synergy with standard genotoxic chemotherapy to kill cancer cells and treat neuroblastoma in mouse models. Amplification of the MYCN oncogene predicts treatment resistance in childhood neuroblastoma. We used a MYC target gene signature that predicts poor neuroblastoma prognosis to identify the histone chaperone FACT (facilitates chromatin transcription) as a crucial mediator of the MYC signal and a therapeutic target in the disease. FACT and MYCN expression created a forward feedback loop in neuroblastoma cells that was essential for maintaining mutual high expression. FACT inhibition by the small-molecule curaxin compound CBL0137 markedly reduced tumor initiation and progression in vivo. CBL0137 exhibited strong synergy with standard chemotherapy by blocking repair of DNA damage caused by genotoxic drugs, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells and suggesting a treatment strategy for MYCN-driven neuroblastoma.

Polyamine pathway inhibition as a novel therapeutic approach to treating neuroblastoma.

Polyamines are highly regulated essential cations that are elevated in rapidly proliferating tissues, including diverse cancers. Expression analyses in neuroblastomas suggest that up-regulation of polyamine pro-synthetic enzymes and down-regulation of catabolic enzymes is associated with poor prognosis. Polyamine sufficiency may be required for MYCN oncogenicity in MYCN amplified neuroblastoma, and targeting polyamine homeostasis may therefore provide an attractive therapeutic approach. ODC1, an oncogenic MYCN target, is rate-limiting for polyamine synthesis, and is overexpressed in many cancers including neuroblastoma. Inhibition of ODC1 by difluoromethylornithine (DFMO) decreased tumor penetrance in TH-MYCN mice treated pre-emptively, and extended survival and synergized with chemotherapy in treating established tumors in both TH-MYCN and xenograft models. Efforts to augment DFMO activity, or otherwise maximally reduce polyamine levels, are focused on antagonizing polyamine uptake or augmenting polyamine export or catabolism. Since polyamine inhibition appears to be clinically well tolerated, these approaches, particularly when combined with chemotherapy, have great potential for improving neuroblastoma outcome in both MYCN amplified and non-MYCN amplified neuroblastomas.

Polyamine antagonist therapies inhibit neuroblastoma initiation and progression

Purpose: Deregulated MYC drives oncogenesis in many tissues yet direct pharmacologic inhibition has proven difficult. MYC coordinately regulates polyamine homeostasis as these essential cations support MYC functions, and drugs that antagonize polyamine sufficiency have synthetic-lethal interactions with MYC. Neuroblastoma is a lethal tumor in which the MYC homologue MYCN, and ODC1, the rate-limiting enzyme in polyamine synthesis, are frequently deregulated so we tested optimized polyamine depletion regimens for activity against neuroblastoma. Experimental Design: We used complementary transgenic and xenograft-bearing neuroblastoma models to assess polyamine antagonists. We investigated difluoromethylornithine (DFMO; an inhibitor of Odc, the rate-limiting enzyme in polyamine synthesis), SAM486 (an inhibitor of Amd1, the second rate-limiting enzyme), and celecoxib (an inducer of Sat1 and polyamine catabolism) in both the preemptive setting and in the treatment of established tumors. In vitro assays were performed to identify mechanisms of activity. Results: An optimized polyamine antagonist regimen using DFMO and SAM486 to inhibit both rate-limiting enzymes in polyamine synthesis potently blocked neuroblastoma initiation in transgenic mice, underscoring the requirement for polyamines in MYC-driven oncogenesis. Furthermore, the combination of DFMO with celecoxib was found to be highly active, alone, and combined with numerous chemotherapy regimens, in regressing established tumors in both models, including tumors harboring highest risk genetic lesions such as MYCN amplification, ALK mutation, and TP53 mutation with multidrug resistance. Conclusions: Given the broad preclinical activity demonstrated by polyamine antagonist regimens across diverse in vivo models, clinical investigation of such approaches in neuroblastoma and potentially other MYC-driven tumors is warranted. Clin Cancer Res; 22(17); 4391–404. ©2016 AACR.

MYCN promotes neuroblastoma malignancy by establishing a regulatory circuit with transcription factor AP4

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4) has been reported to be a direct transcriptional target of MYC. We show for the first time that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome. siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells led to inhibition of cell proliferation and migration. Chromatin immunoprecipitation assay demonstrated that TFAP4 expression is positively regulated by MYCN. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study suggests a regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying potential therapeutic targets for aggressive forms of this disease.

Abstract 2450: MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis

Amplification of the MYCN oncogene, a member of the MYC family of transcriptional regulators, is one of the most powerful prognostic markers identified for poor outcome in neuroblastoma, the most common extracranial solid cancer in childhood. While MYCN has been established as a key driver of malignancy in neuroblastoma, the underlying molecular mechanisms are poorly understood. Transcription factor activating enhancer binding protein-4 (TFAP4), which plays important roles in cancer progression, has been reported to be a direct transcriptional target of MYC. In this study, we have shown that high expression of TFAP4 in primary neuroblastoma patients is associated with poor clinical outcome and furthermore that siRNA-mediated suppression of TFAP4 in MYCN-expressing neuroblastoma cells impaired migration and colony formation, and led to an increased proportion of cells in G1/S phase of the cell cycle. Chromatin immunoprecipitation and luciferase reporter assays demonstrated that TFAP4 expression is positively regulated by MYCN through direct promoter binding. In addition, when MYCN was overexpressed in neuroblastoma cells, TFAP4 was required for the observed increase in cell migration. Microarray analysis identified genes regulated by both MYCN and TFAP4 in neuroblastoma cells, including Phosphoribosyl-pyrophosphate synthetase-2 (PRPS2) and Syndecan-1 (SDC1), which are involved in cancer cell proliferation and metastasis. Overall this study unveils a complex regulatory circuit in which MYCN by elevating TFAP4 expression, cooperates with it to control a specific set of genes involved in tumor progression. These findings highlight the existence of a MYCN-TFAP4 axis in MYCN-driven neuroblastoma as well as identifying relevant therapeutic targets for aggressive forms of this disease. Citation Format: Chengyuan Xue, Denise M. Yu, Samuele Gherardi, Jessica Koach, Giorgio Milazzo, Laura Gamble, Bing Liu, Amanda Russell, Tao Liu, Belamy B. Cheung, Glenn M. Marshall, Giovanni Perini, Michelle Haber, Murray D. Norris. MYCN and TFAP4 promote neuroblastoma malignancy by cooperating in the regulation a subset of target genes involved in cancer cell growth and metastasis. [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 2450.

Abstract 2006: AMX-513 polyamine depletion therapy inhibits tumor growth and reverses immunosuppression in cancers including MYC-driven neuroblastoma and pancreatic cancer

Tumorigenesis is associated with increased polyamine levels and involves the induction of ornithine decarboxylase (ODC), the initial rate-limiting enzyme in polyamine biosynthesis, and increased uptake of polyamines from the blood and diet. As well as contributing to proliferation, polyamines are reported to exert an immunosuppressive effect. Amplification of the MYC/MYCN oncogenes has been shown to directly induce ODC activity and inhibition of this enzyme by α-difluoromethylornithine (DFMO) markedly delays tumor development. Aminex Therapeutics is developing a polyamine depletion approach targeting both biosynthesis and transport of polyamines with AMX-513, a combination of the approved ODC inhibitor, DFMO, together with AMXT 1501, an alkylated polyamine mimetic which blocks polyamine uptake. In the syngeneic CT26.CL25 mouse model of colorectal cancer, AMX-513, dosed daily for four weeks, reduced tumor growth > 75% compared to vehicle-treated control in immunocompetent Balb/C mice. There was no effect in athymic nude mice indicating that tumor growth inhibition by AMX-513 is T-cell-dependent. In the induced transgenic K6/ODC squamous tumor mouse model, stable regression was sustained 10 weeks after treatment ended and was accompanied by tumor infiltrate increases in IFNγ and in CD3+ and CD8+ T-cells. Tumor infiltrates from AMX-513-treated KPC pancreatic cancer transgenic mice with tumor regressions showed >90% reductions in myeloid-derived suppressor cells (MDSCs; CD11b+ Gr-1+) but no changes in mature myeloid cells (CD11+Gr-1neg) by FACS analysis. AMX-513 treatment did not impact the percentage or number of CD4+CD25+FoxP3+ Tregs, but did significantly increase the percentage of activated CD8+ T cells in tumors. Neuroblastoma is an aggressive childhood cancer frequently associated with MYCN and ODC deregulation. In neuroblastoma cell lines, the AMX-513 combination was highly synergistic (CI In conclusion, AMX-513 treatment alone or in combination with other cancer therapies results in significant tumor growth reduction in multiple cancer models and demonstrates novel immunotherapeutic potential. Clinical evaluation of AMX-513 is planned in 2017. Citation Format: Mark R. Burns, Kathy Fosnaugh, Michael G. Palfreyman, Laura Gamble, Jayne Murray, Sophie Allan, Georgina Eden, Sara Sarraf, Murray Norris, David Ziegler, Michelle Haber. AMX-513 polyamine depletion therapy inhibits tumor growth and reverses immunosuppression in cancers including MYC-driven neuroblastoma and pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2017-2006

Abstract PR09: MYCN and is a therapeutic target in neuroblastoma

Neuroblastoma is a childhood cancer of the sympathetic nervous system. Approximately 20% of patients present with an aggressive metastatic disease characterized by amplification of the MYCN locus. However, an additional 30% of patients suffer from an equally aggressive disease, yet there are no unified genetic markers available. To identify potential biomarkers of poor prognosis in neuroblastoma, we conducted unsupervised hierarchical clustering on 649 primary neuroblastoma tumors based on a 51 gene MYC core target signature. This demonstrated strong activation within the MYCN amplified cohort as expected, but surprisingly there was a large subset of MYCN non-amplified patients that exhibited high MYC signaling. High expression of this MYC signature proved to be strongly predictive of poor overall survival, in both the full cohort (Hazard ratio (HR): 47.6, p= 2.7x10-35, n=649) and in MYCN non-amplified patients (HR=32.8, p=1.3x10-13, n=554), suggesting that neuroblastoma can be driven by MYC signaling in the absence of MYCN amplification. To evaluate potential therapeutic targets within this MYC signature, we conducted cox regression analysis on the 51 individual members and identified that SPT16 was a particularly potent predictor of poor outcome in these 649 neuroblastoma patients (HR: 7.2, p=2.7x10-17). SPT16 is a subunit of the FACT histone chaperone complex, which together with its heterodimer partner SSRP1, has previously been shown to function in dynamic regulation of chromatin to drive transcription, DNA replication and DNA repair. Similar to SPT16, high SSRP1 expression was a strong predictor of poor prognosis in 649 neuroblastoma patients (HR: 12.5, p=5.812x10-26) implicating FACT as a mediator of MYC driven neuroblastoma. Next we investigated the biological relationship between FACT and MYCN in neuroblastoma. siRNA experiments showed that FACT and MYCN participate in a positive feedback transcriptional loop that is essential for maintaining mutual high expression. Immunohistochemistry in sympathetic ganglia of pre-tumor TH-MYCN mouse model of neuroblastoma identified that FACT was highly expressed at tumor initiation and chemical inhibition of FACT by CBL0137 markedly reduced tumor initiation and subsequent tumorigenesis in vivo. Next we tested CBL0137 against established neuroblastoma in TH-MYCN mice. CBL0137 was highly cytotoxic to tumors either as a single agent or by synergistically promoting clinically used chemotherapeutics. This was accompanied by marked MYCN depletion and prolonged tumor regression in a majority of recipient mice. Pulsed-field electrophoresis showed that CBL0137 impaired FACT dependent DNA damage repair in the presence of genotoxic chemotherapy, thus creating a synthetic lethal environment in MYCN-amplified neuroblastoma cells. Together our data define FACT as a mediator of neuroblastoma tumorigenesis and MYCN-related treatment target. Given the current poor survival rates of children with high-risk neuroblastoma, our findings provide support for the clinical development of CBL0137 as a novel treatment approach for this refractory malignancy. Citation Format: Daniel R. Carter, Jayne Murray, Belamy B. Cheung, Heyam Kalla, Laura Gamble, Joanna Tsang, Selina Sutton, Jessica Koach, Sarah Syed, Andrew Gifford, Natalia Issaeva, Asel Biktasova, Bernard Atmadibrata, Yuting Sun, Nicolas Sokolowski, Dora Ling, Patrick Y. Kim, Hannah Webber, Ashleigh Clark, Michelle Ruhle, Bing Liu, Andre Oberthuer, Matthias Fischer, Jennifer Byrne, Federica Saletta, Andrei Purmal, David Ziegler, Tao Liu, Katerina V. Gurova, Andrei V. Gudkov, Murray D. Norris, Michelle Haber, Glenn M. Marshall. MYCN and is a therapeutic target in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Myc: From Biology to Therapy; Jan 7-10, 2015; La Jolla, CA. Philadelphia (PA): AACR; Mol Cancer Res 2015;13(10 Suppl):Abstract nr PR09.

Abstract 1611: The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting

Background: Despite the success of chemotherapy in improving the overall survival rate of childhood cancer, a number of types of children9s cancers still have dismal outcomes. Included here are high risk neuroblastomas, Diffuse Intrinsic Pontine Gliomas (DIPG), and infant leukemias with MLL translocations. New treatments for these aggressive childhood cancers are urgently needed. Evidence is emerging of the importance of alterations in chromatin modifier genes in pediatric cancers. In this regard, CBL0137 is a carbazole-based anti-cancer agent with a unique mechanism of action. It is an indirect inhibitor of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). Inhibition of FACT by CBL0137 modulates the activity of several transcription factors involved in cancer: NF-kB and HSF1 are suppressed, while p53 is activated (Science Transl Med, 2011). We have examined FACT expression in neuroblastoma, DIPG and MLL leukemia, as well as the efficacy of CBL0137 in preclinical models of these diseases. Methods: Expression of the FACT subunits, SSRP1 and SPT16, was examined in neuroblastoma, DIPG and MLL leukemia cells using RT-PCR and Western analysis. The clinical significance of SSRP1 and SPT16 was also analysed using expression array data on 650 primary untreated neuroblastomas. Colony-forming assays were used to study the effect of CBL0137, either alone or combined with chemotherapeutic drugs. Cohorts of neuroblastoma, DIPG and MLL leukemia xenografted mice, as well as neuroblastoma-prone TH-MYCN mice, were treated with CBL0137, alone or combined with chemotherapeutic drugs. Results: High levels of SSRP1 and SPT16 expression were observed in all three types of child cancer. In addition, in neuroblastoma, the two FACT subunits were associated with MYCN amplification, and were strongly predictive of poor outcome (p Conclusions: Targeting FACT offers a highly promising novel therapeutic approach for aggressive childhood cancers. The results for CBL0137 are as good or better than any chemotherapy regimens we have tested in our preclinical models, and a Phase I COG trial of this nongenotoxic agent in refractory pediatric cancer patients is currently being planned. Citation Format: Michelle Haber, Jayne Murray, Laura Gamble, Ashleigh Carnegie-Clark, Hannah Webber, Michelle Ruhle, Michelle J. Henderson, Shiloh Middlemass, Daniel Carter, Maria Tsoli, Anahid Ehteda, Sandy Simon, Andre Oberthuer, Matthias Fischer, Katerina Gurova, Catherine Burkhart, Andrei Purmal, Richard B. Lock, David Ziegler, Glenn M. Marshall, Andrei V. Gudkov, Murray D. Norris. The FACT histone chaperone complex is highly expressed in aggressive drug refractory childhood cancers and the anti-FACT compound CBL0137 represents a highly promising therapeutic approach in this setting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1611. doi:10.1158/1538-7445.AM2015-1611