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HT10806TG_CBL0137 treatment

Supplementary Figure Legends S1-S16

HT10806TG_Combination treatment

HT10806TG_Control

Purpose:We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma.Experimental Design:The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction.Results:The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination.Conclusions:The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

Supplementary Figures S1-S16

HT10806TG_Panobinostat treatment

Supplementary Tables S1-S5

Purpose: We investigated whether targeting chromatin stability through a combination of the curaxin CBL0137 with the histone deacetylase (HDAC) inhibitor, panobinostat, constitutes an effective multimodal treatment for high-risk neuroblastoma. Experimental Design: The effects of the drug combination on cancer growth were examined in vitro and in animal models of MYCN-amplified neuroblastoma. The molecular mechanisms of action were analyzed by multiple techniques including whole transcriptome profiling, immune deconvolution analysis, immunofluorescence, flow cytometry, pulsed-field gel electrophoresis, assays to assess cell growth and apoptosis, and a range of cell-based reporter systems to examine histone eviction, heterochromatin transcription, and chromatin compaction. Results: The combination of CBL0137 and panobinostat enhanced nucleosome destabilization, induced an IFN response, inhibited DNA damage repair, and synergistically suppressed cancer cell growth. Similar synergistic effects were observed when combining CBL0137 with other HDAC inhibitors. The CBL0137/panobinostat combination significantly delayed cancer progression in xenograft models of poor outcome high-risk neuroblastoma. Complete tumor regression was achieved in the transgenic Th-MYCN neuroblastoma model which was accompanied by induction of a type I IFN and immune response. Tumor transplantation experiments further confirmed that the presence of a competent adaptive immune system component allowed the exploitation of the full potential of the drug combination. Conclusions: The combination of CBL0137 and panobinostat is effective and well-tolerated in preclinical models of aggressive high-risk neuroblastoma, warranting further preclinical and clinical investigation in other pediatric cancers. On the basis of its potential to boost IFN and immune responses in cancer models, the drug combination holds promising potential for addition to immunotherapies.

Despite the increase in overall child cancer survival rates, pediatric malignancies such as high-risk neuroblastoma, high-risk leukemias (including MLL-translocated infant ALL), and aggressive brain tumors (including DIPG) remain refractory to current multimodal therapies. We have been developing new treatment approaches for these aggressive childhood cancers by (i) utilizing novel targeted therapies either alone or combined with other new agents or established chemotherapeutic drugs, and (ii) by developing new drugs that target key pathways in these child cancers. In neuroblastoma, we have targeted polyamines, showing that combined inhibition of polyamine synthesis by the ODC1 inhibitor DFMO, and of polyamine uptake using the small-molecule drug AMXT 1501, is highly effective at inhibiting tumor growth in Th-MYCN transgenic mice. This combination also shows great efficacy in preclinical models of DIPG, and clinical trials for these diseases are now being planned. We are also targeting metabolism of arginine, the precursor of ornithine, using the pegylated-recombinant arginase BCT-100, which significantly delays tumor development and prolongs survival of neuroblastoma-prone Th-MYCN mice. We have further shown that combining BCT-100 with either DFMO or conventional chemotherapy results in increased survival benefit. CBL0137 is a nontoxic novel anticancer drug currently in phase I trial for adult refractory and relapsed cancers. CBL0137 destabilizes nucleosomes and traps histone chaperone FACT into chromatin, thereby modulating several anticancer mechanisms. We have shown that CBL0137 is effective in mouse models of neuroblastoma, MLL-rearranged leukemia, and DIPG, and that its action is potentiated by the HDAC inhibitor, panobinostat. Moreover, we have developed OT-82, a novel nontoxic NAMPT inhibitor with impressive anticancer activity against mouse models of high-risk childhood ALL, potentiating standard-of-care drugs, and showing similar efficacy as the three-drug induction-type treatment used for pediatric ALL. In addition, for all Australian children with high-risk malignancies, we have developed the Zero Childhood Cancer national precision medicine program. ZERO utilizes whole-genome and whole-transcriptome sequencing, methylation profiling, and where possible, in vitro and in vivo drug testing. To date (July 2019), 74% of 207 patients on the national clinical trial have received a Multidisciplinary Tumor Board recommendation (therapy, germline referral, or change of diagnosis), and of 25 patients with evaluable response data thus far who have received the ZERO recommended therapy, a significant proportion have had a complete response, partial response, or maintained stable disease. Moreover, early experience with drug efficacy studies suggests these data may corroborate genomic therapeutic recommendations and may also identify unanticipated active therapeutics. Citation Format: Michelle Haber, Laura Gamble, Lin Xiao, Ruby Pandher, Klaartje Somers, Jayne Murray, Aaminah Khan, Denise Yu, Laura Franshaw, Mark R. Burns, Maria Tsoli, Anahid Ehteda, Anthony Cesare, Aisling O’Connor, Francis Mussai, Carmela de Santo, Paul Cheng, Lioubov Korotchkina, Katerina Gurova, Vanessa Tyrrell, Emily Mould, Loretta Lau, Dong Anh Khuong Quang, Chelsea Mayoh, Greg Arndt, Paulette Barahona, Tim Failes, Jamie Fletcher, Noemi Fuentes- Bolanos, Marie-Emilie Gauthier, Andrew Gifford, Dylan Grebert-Wade, Alvin Kamili, Amit Kumar, Sumanth Nagabushan, Tracey O’Brien, Patrick Strong, Alexandra Sherstyuk, David Thomas, Toby Trahair, Katherine Tucker, Meera Warby, Marie Wong, Jinhan Xie, Kathryn Evans, Richard Lock, Olga B. Chernova, Michelle Henderson, Andrei V Gudkov, Paul Ekert, Mark J. Cowley, Glenn M. Marshall, David S. Ziegler, Murray D. Norris. Molecular targeted therapies and precision medicine for children with neuroblastoma and other refractory malignancies [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr IA13.

In human somatic cells, telomeres shorten every cell division due to the end replication problem. Once reaching a critically short length, the cells will undergo permanent cell cycle arrest and become senescent. Cancer cells acquire unlimited proliferation ability by activation of a telomere maintenance mechanism, either the enzyme telomerase or the homologous recombination-based mechanism Alternative Lengthening of Telomeres (ALT). Cancers that utilize the ALT mechanism commonly are deficient for ATRX protein expression, are difficult to treat, and have a poor prognosis. We discovered that ICP0-null herpes simplex virus type 1 (HSV-1) was ten to one thousand-fold more effective in killing cancer cell lines that are ATRX-deficient. Sensitivity to mutant HSV-1 infection resulted from ATRX-dependent regulation of PML expression at both the transcriptional and post-transcriptional levels. Reduction of PML protein resulted in a concomitant reduction in PML nuclear bodies, which weakened the innate cellular immunity to viral infection. Infection of co-cultured primary and ATRX-null cancer cells revealed that mutant ICP0-null HSV-1 treatment preferentially killed ATRX-null cells. Our results suggest that mutant ICP0-null HSV-1 may have therapeutic benefit against ATRX-null cells. Moreover, these data provide an applicable approach for predicting, based on tumor ATRX or PML protein status, which tumors will respond to an oncolytic herpes virus. Citation Format: Mingqi Han, Christine E. Napier, Sonja Frölich, Roger D. Everett, Anthony J. Cesare, Roger R. Reddel. Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A22.

Telomere shortening and disruption of telomeric components are pathways that induce telomere deprotection. Here we describe another pathway, where prolonged mitotic arrest induces damage signals at telomeres. Exposure to microtubule drugs, kinesin inhibitors, proteasome inhibitors or the disruption of proper chromosome alignment resulted in the formation of damage-foci at telomeres. Induction of mitotic telomere deprotection coincided with dissociation of TRF2 from telomeres, telomeric 3′-overhang degradation and ATM activation, and could be suppressed by TRF2 overexpression or inhibition of Aurora B kinase. Normal cells that escape from prolonged mitotic arrest halted in the following G1 phase, whereas cells lacking p53 continued to cycle and became aneuploid. We propose a telomere dependent mitotic duration checkpoint that ensures elimination of cells, which fail to progress through mitosis. To avoid unwanted checkpoint activation by natural chromosome ends, cells have evolved telomeres. Human telomeres are composed of double stranded TTAGGG repeats and a single stranded G rich 3′ overhang, which are covered and protected by shelterin. Among the six shelterin components TRF2 and POT1 have predominantly been implicated in chromosome end protection by preventing ATM- and ATR-dependent checkpoint activation. Upon disruption of TRF2 or POT1 telomeres are recognized as sites of DNA damage, resulting in phosphorylation of histone H2AX (γ-H2AX) within the telomeric and sub-telomeric chromatin and association of 53BP1 with the chromosome ends. The co-localization of DNA-damage response factors and chromosome ends can be visualized as telomere dysfunction-induced foci (TIF). TIF have also been intimately linked to replicative senescence and shown to occur spontaneously in cancer cell lines. Cells arrested in mitosis are known to either die during mitotic arrest, or skip cytokinesis and “slip” into the subsequent G1 phase of the cell cycle. Mitotic slippage occurs through the degradation of Cyclin B1 in the presence of the active spindle assembly checkpoint (SAC). Cells that exit from prolonged mitotic arrest or progress through mitotic slippage exhibit various fates, including apoptosis or p53-dependent cell cycle arrest. In both normal and cancer cells, cell death during mitotic arrest, or apoptosis or senescence after escape from prolonged mitotic arrest are crucial for preventing chromosome instability. A failure to remove cells from the cycling population following prolonged mitotic arrest may allow cells to continue propagating with an abnormal number of chromosomes. However, despite intense research, the molecular mechanisms that trigger growth arrest or death in mitotically arrested cultures have not yet been identified. In the course of experimentation to explore putative telomeric functions for cohesin, we found that mitotic arrest per se induces telomere deprotection in primary and transformed human cells. Telomere deprotection during mitotic arrest associated with loss of the telomeric 3′-overhangs, led to ATM activation and was ATM dependent. TRF2 was dissociated from telomeres during prolonged mitotic arrest, providing the molecular basis for overhang loss and ATM activation, which was emphasized by the finding that TRF2 overexpression protected telomeres from the damage machinery during mitotic arrest. Inhibition of Aurora B kinase suppressed the telomere deprotection phenotype, suggesting the involvment of the SAC. Cells suffering from mitotic telomere deprotection underwent p53 dependent cell cycle arrest in the following G1 phase after mitotic release, while cells lacking p53 function continued to cycle and became aneuploid. Our findings provide a molecular mechanism explaining the induction of DNA damage signaling, cell cycle arrest or apoptosis following prolonged mitotic arrest, and explain the mechanism of action of therapeutic drugs, such as Taxol, Vinblastine and Velcade, which all inhibit mitotic progression. We propose that telomeric destabilization during mitotic arrest induces DNA damage signaling and serves as a mitotic duration checkpoint, responsible for eliminating cells that fail to progress through mitosis properly. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY23-02. doi:1538-7445.AM2012-SY23-02