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RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) is tightly regulated downstream of oncogenic pathways, and its dysregulation is a common feature in cancer. We evaluated CX-5461, the first-in-class selective rDNA transcription inhibitor, in a first-in-human, phase I dose-escalation study in advanced hematologic cancers. Administration of CX-5461 intravenously once every 3 weeks to 5 cohorts determined an MTD of 170 mg/m2, with a predictable pharmacokinetic profile. The dose-limiting toxicity was palmar–plantar erythrodysesthesia; photosensitivity was a dose-independent adverse event (AE), manageable by preventive measures. CX-5461 induced rapid on-target inhibition of rDNA transcription, with p53 activation detected in tumor cells from one patient achieving a clinical response. One patient with anaplastic large cell lymphoma attained a prolonged partial response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable disease as best response. CX-5461 is safe at doses associated with clinical benefit and dermatologic AEs are manageable.Significance:CX-5461 is a first-in-class selective inhibitor of rDNA transcription. This first-in-human study establishes the feasibility of targeting this process, demonstrating single-agent antitumor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profile allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies.This article is highlighted in the In This Issue feature, p. 983

This figure shows supplementary data pertinent to Figure 1 including cell death, cell signalling and mRNA transcription analysis data.

PDF file - 209K, Everolimus is associated with changes to the immunophenotype. SF8: Continued daily dosing with everolimus results in cell cycle arrest

PDF file - 1.1MB, Quantitation of SA-B-gal and immunostaining. SF10: Markers of senescence. SF11: Overall survival of mice transplanted with a p53 mutant lymphoma

Exposure-response relationship between CX-5461 levels and rDNA transcription rate in normal PBMCs

PDF file - 28K, Primer sequences. ST2: Baseline characteristics of randomized mice

All Supplementary Tables

PDF file - 165K, Morphology of tumors. SF2: Everolimus restores B-cell development. SF3: B-cell precursors in the bone marrow

PDF file - 121K, Full lenght Western blots

PDF file - 2.3MB, Bone marrow cellularity. SF5: Heightened sensitivity of B-cells. SF6. Survival benefit of everolimus

Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease., Acquired resistance limits the efficacy of PARP inhibitors (PARPi) in high grade serous ovarian cancer (HGSOC). Here, the authors show that inhibition of RNA polymerase I transcription using CX-5461 increases the therapeutic efficacy of PARPi and overcomes PARPi resistance in PDX models of HGSOC.

Significance Targeting CDK4/6 shows great promise in the treatment of many solid cancers, including melanoma. This study has uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. In melanoma, palbociclib activates p53 via modulating PRMT5-dependent alternate MDM4 pre-mRNA splicing, which results in a robust decrease in MDM4 protein expression. Loss of palbociclib regulation of the PRMT5–MDM4 axis leads to drug resistance. Dual inhibition of CDK4/6 and PRMT5 potently suppresses melanoma tumor growth and is well tolerated in vivo. Our findings not only have immediate implications for the advancement of CDK4/6 inhibition for treating melanoma, but also more generally offer insights into CDK4/6 mechanism of action., Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5–MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5–MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.

RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) is tightly regulated downstream of oncogenic pathways, and its dysregulation is a common feature in cancer. We evaluated CX-5461, the first-in-class selective rDNA transcription inhibitor, in a first-in-human, phase I dose-escalation study in advanced hematologic cancers. Administration of CX-5461 intravenously once every 3 weeks to 5 cohorts determined an MTD of 170 mg/m2, with a predictable pharmacokinetic profile. The dose-limiting toxicity was palmar–plantar erythrodysesthesia; photosensitivity was a dose-independent adverse event (AE), manageable by preventive measures. CX-5461 induced rapid on-target inhibition of rDNA transcription, with p53 activation detected in tumor cells from one patient achieving a clinical response. One patient with anaplastic large cell lymphoma attained a prolonged partial response and 5 patients with myeloma and diffuse large B-cell lymphoma achieved stable disease as best response. CX-5461 is safe at doses associated with clinical benefit and dermatologic AEs are manageable. Significance: CX-5461 is a first-in-class selective inhibitor of rDNA transcription. This first-in-human study establishes the feasibility of targeting this process, demonstrating single-agent antitumor activity against advanced hematologic cancers with predictable pharmacokinetics and a safety profile allowing prolonged dosing. Consistent with preclinical data, antitumor activity was observed in TP53 wild-type and mutant malignancies. This article is highlighted in the In This Issue feature, p. 983

The three-dimensional organization of the genome contributes to its maintenance and regulation. While chromosomal regions associate with nucleolar ribosomal RNA genes (rDNA), the biological significance of rDNA-genome interactions and whether they are dynamically regulated during disease remain unclear. rDNA chromatin exists in multiple inactive and active states and their transition is regulated by the RNA polymerase I transcription factor UBTF. Here, using a MYC-driven lymphoma model, we demonstrate that during malignant progression the rDNA chromatin converts to the open state, which is required for tumor cell survival. Moreover, this rDNA transition co-occurs with a reorganization of rDNA-genome contacts which correlate with gene expression changes at associated loci, impacting gene ontologies including B-cell differentiation, cell growth and metabolism. We propose that UBTF-mediated conversion to open rDNA chromatin during malignant transformation contributes to the regulation of specific gene pathways that regulate growth and differentiation through reformed long-range physical interactions with the rDNA., Jeannine Diesch et al. report the changes in rDNA chromatin state associated with cell transition into malignancy. They show that a specific transcription factor regulates this transition by altering rDNA chromatin, resulting in the reorganization of contacts between rDNA and the genome.

Elevated ribosome biogenesis in oncogene‐driven cancers is commonly targeted by DNA‐damaging cytotoxic drugs. Our previous first‐in‐human trial of CX‐5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA polymerase I (Pol I) transcription, revealed single‐agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in the in vivo efficacy of CX‐5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here, we reveal the molecular basis for this improved efficacy observed in vivo, which is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this cotreatment is driven by translational rewiring that results in dysregulated cellular metabolism and induction of a cAMP‐dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies thus identify key molecular mechanisms underpinning the response of blood cancers to selective inhibition of ribosome biogenesis and define metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I‐directed therapies., The elevated metabolic activity and activation of cAMP‐dependent pro‐survival mechanisms that lead to resistance of hematological cancers towards rRNA synthesis inhibition also poses a potentially targetable vulnerability.

Elevated ribosome biogenesis in oncogene-driven cancers is commonly targeted by DNA-damaging cytotoxic drugs. Our first-in-human trial of CX-5461, a novel, less genotoxic agent that specifically inhibits ribosome biogenesis via suppression of RNA Polymerase I (Pol I) transcription, revealed single agent efficacy in refractory blood cancers. Despite this clinical response, patients were not cured. In parallel, we demonstrated a marked improvement in thein vivoefficacy of CX-5461 in combination with PI3K/AKT/mTORC1 pathway inhibitors. Here we show that this improved efficacy is associated with specific suppression of translation of mRNAs encoding regulators of cellular metabolism. Importantly, acquired resistance to this co-treatment is driven by translational re-wiring that results in dysregulated cellular metabolism and induction of a cAMP-dependent pathway critical for the survival of blood cancers including lymphoma and acute myeloid leukemia. Our studies identify the molecular mechanisms underpinning the response of blood cancers to selective ribosome biogenesis inhibitors and identify metabolic vulnerabilities that will facilitate the rational design of more effective regimens for Pol I-directed therapies.

High-grade serous ovarian cancer (HGSOC) accounts for the majority of ovarian cancer and has a dismal prognosis. PARP inhibitors (PARPi) have revolutionized disease management of patients with homologous recombination (HR) DNA repair-deficient HGSOC. However, acquired resistance to PARPi by complex mechanisms including HR restoration and stabilisation of replication forks is a major challenge in the clinic. Here, we demonstrate CX-5461, an inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress at rDNA leading to activation of DNA damage response and DNA damage involving MRE11-dependent degradation of replication forks. CX-5461 cooperates with PARPi in exacerbating DNA damage and enhances synthetic lethal interactions of PARPi with HR deficiency in HGSOC-patient-derived xenograft (PDX)in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi and destabilises replication forks irrespective of HR pathway status, overcoming two well-known mechanisms of resistance to PARPi. Importantly, CX-5461 exhibits single agent efficacy in PARPi-resistant HGSOC-PDX. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Therefore, CX-5461 is a promising therapy alone and in combination therapy with PARPi in HR-deficient HGSOC. CX-5461 is also an exciting treatment option for patients with relapsed HGSOC tumors that have poor clinical outcome.

RNA polymerase I (Pol I)-mediated transcription of the ribosomal RNA genes (rDNA) is confined to the nucleolus and is a rate-limiting step for cell growth and proliferation. Inhibition of Pol I by CX-5461 can selectively induce p53-mediated apoptosis of tumour cells in vivo. Currently, CX-5461 is in clinical trial for patients with advanced haematological malignancies (Peter Mac, Melbourne). Here we demonstrate that CX-5461 also induces p53-independent cell cycle checkpoints mediated by ATM/ATR signaling in the absence of DNA damage. Further, our data demonstrate that the combination of drugs targeting ATM/ATR signaling and CX-5461 leads to enhanced therapeutic benefit in treating p53-null tumours in vivo, which are normally refractory to each drug alone. Mechanistically, we show that CX-5461 induces an unusual chromatin structure in which transcriptionally competent relaxed rDNA repeats are devoid of transcribing Pol I leading to activation of ATM signaling within the nucleoli. Thus, we propose that acute inhibition of Pol transcription initiation by CX-5461 induces a novel nucleolar stress response that can be targeted to improve therapeutic efficacy.

Despite two decades of research, the major function of FBP-family KH domain proteins during animal development remains controversial. The literature is divided between RNA processing and transcriptional functions for these single stranded nucleic acid binding proteins. Using Drosophila, where the three mammalian FBP proteins (FBP1-3) are represented by one ortholog, Psi, we demonstrate the primary developmental role is control of cell and tissue growth. Co-IP-mass spectrometry positioned Psi in an interactome predominantly comprised of RNA Polymerase II (RNA Pol II) transcriptional machinery and we demonstrate Psi is a potent transcriptional activator. The most striking interaction was between Psi and the transcriptional mediator (MED) complex, a known sensor of signaling inputs. Moreover, genetic manipulation of MED activity modified Psi-dependent growth, which suggests Psi interacts with MED to integrate developmental growth signals. Our data suggest the key target of the Psi/MED network in controlling developmentally regulated tissue growth is the transcription factor MYC. As FBP1 has been implicated in controlling expression of the MYC oncogene, we predict interaction between MED and FBP1 might also have implications for cancer initiation and progression.

Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I–mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K–AKT–mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eμ-Myc lymphoma–bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. Significance: Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eμ-Myc lymphoma–bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer. Cancer Discov; 6(1); 59–70. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 1

Introduction: PARP inhibitors (PARPi) have revolutionized disease management of patients with homologous recombination (HR) DNA repair-deficient high-grade serous ovarian cancer (HGSOC). However, acquired resistance to PARPi is a major challenge in the clinic. The specific inhibitor of RNA polymerase I (Pol I) transcription of ribosomal RNA genes (rDNA) has demonstrated single-agent antitumor activity in p53 wild-type and p53-mutant hematologic malignancies (first-in-human trial, dose escalation study of CX-5461 at Peter MacCallum Cancer Centre) (Khot et al., Cancer Discov 2019). CX-5461 has also been reported to exhibit synthetic lethality with BRCA1/2 deficiency through stabilization of G-quadruplex DNA (GQ) structures. Here, we investigate the efficacy of CX-5461 in treating HGSOC. Experimental Design: The mechanisms by which CX-5461 induces DNA damage response (DDR) and displays synthetic lethality in HR-deficient HGSOC cells are explored. We present in vivo data of mice bearing two functionally and genomically profiled HGSOC-patient-derived xenograft (PDX)s treated with CX-5461 and olaparib, alone and in combination. We also investigate CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Results: Utilizing ovarian cancer cell lines, we demonstrate that sensitivity to CX-5461 is associated with “BRCA1 mutation” and “MYC targets” gene expression signatures. In addition, sensitivity to CX-5461 is associated with high basal rates of Pol I transcription. Importantly, we demonstrate a novel mechanism for CX-5461 synthetic lethal interaction with HR deficiency mediated through the induction of replication stress at rDNA repeats. Our data reveal CX-5461-mediated DDR in HR-deficient cells does not involve stabilization of GQ structures as previously proposed. On the contrary, we show definitively that CX-5461 inhibits Pol I recruitment leading to rDNA chromatin defects including stabilization of R-loops, single-stranded DNA, and replication stress at the rDNA. Mechanistically, we demonstrate CX-5461 leads to replication-dependent DNA damage involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 has a different sensitivity spectrum to olaparib and cooperates with PARPi in exacerbating replication stress, leading to enhanced therapeutic efficacy in HR-deficient HGSOC-PDX in vivo compared to single-agent treatment of both drugs. Further, CX-5461 exhibits single-agent efficacy in olaparib-resistant HGSOC-PDX overcoming PARPi-resistance mechanisms involving fork protection. Importantly, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. Conclusions: CX-5461 is a promising therapy alone and in combination therapy with PARPi in HR-deficient HGSOC. CX-5461 also has exciting potential as a treatment option for patients with relapsed HGSOC tumors that have high MYC activity and poor clinical outcome; these patients currently have very limited effective treatment options. This abstract is also being presented as Poster A71. Citation Format: Elaine Sanij, Katherine Hannan, Jiachen Xuan, Shunfei Yan, Jessica A. Ahern, Anna S. Trigos, Natalie Brajanovski, Jinbae Son, Keefe T. Chan, Olga Kondrashova, Elizabeth Lieschke, Matthew J. Wakefield, Sarah Ellis, Carleen Cullinane, Gretchen Poortinga, Kum Kum Khanna, Linda Mileshkin, Grant A. McArthur, John Soong, Els M. Berns, Ross D. Hannan, Clare L. Scott, Karen E. Sheppard, Richard B. Pearson. Inhibition of RNA polymerase I transcription activates targeted DNA damage response and enhances the efficacy of PARP inhibitors in high-grade serous ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr PR13.

Background: The ribosomal biogenesis inhibitor CX-5461 is a first-in-class selective inhibitor of ribosomal RNA gene transcription with single-agent antitumor activity against advanced hematologic cancers and synergistic activity when combined with front-line and emerging agents (Devlin et al. 2015, Sornkom et al. 2018, Maclachlan et al. 2018). It has predictable pharmacokinetics and a safety profile allowing prolonged dosing (Khot et al, Cancer Discov 2019). Unexpectedly, CX-5461 treatment increases platelet count by ~50% in tumour diseased mice; an effect that was maintained for up to 6 months of treatment. Aims: We investigated mechanisms that may contribute to increased platelet counts following CX-5461 treatment in a liver carcinoma mouse model or disease-free mice and evaluated blood test data from patients with haematological malignancies treated with CX-5461. Methods: We evaluated blood cell numbers in blood from vehicle- or CX-5461 treated mice with liver carcinoma, and blood cell numbers, bone marrow megakaryocytes, platelet receptors, immature platelets, platelet function, thrombopoietin (TPO) and inflammatory cytokine levels in disease-free C57BL/6 KaLwRij mice treated with 35 mg/kg CX-5461 or vehicle (n=15). Human platelet receptors and function were assessed in CX-5461-treated platelet-rich plasma by flow cytometry, platelet spreading assays and light transmission aggregometry. We evaluated temporal platelet count, and the platelet activation marker soluble glycoprotein (GP) VI in patient plasma samples who had received a single dose of 25-250 mg/m2 CX-5461 (n=16) with 1-way ANOVA. Results: In a model of liver carcinoma, mice with disease displayed significantly elevated (2.3-fold) platelet counts after 3 months treatment with biweekly injections of 35 mg/kg CX-5461 compared with vehicle-treated diseased mice (p = 0.0013). Disease-free mice treated for 7 or 14 days (3 or 6 x 35 mg/kg doses) with CX-5461 showed ~60% increase in platelet count; in comparison white and red cells were mildly suppressed. The CX-5461-mediated platelet increase at d7 (p Conclusions: CX-5461 treatment of mice for two weeks increased platelet counts, megakaryocyte number and immature platelet fraction by an unknown mechanism however IL-6 has been reported by others to enhance megakaryopoeisis. Brief CX-5461 treatment in a group of patients with advanced haematological malignancy resulted in small increases in platelet count with retained platelet activation and treatment ex-vivo demonstrated platelet function to be unaffected. Future work will explore the link between CX-5461 treatment and megakaryopoiesis and thrombopoiesis, including the potential for CX-5461 to ameliorate drug-induced thrombocytopenia. Disclosures Harrison: Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GSK: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: investigator on studies, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hannan:Pimera: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity). Refereed/Peer-reviewed

Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naive, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors.

Trypanosoma brucei relies on an essential Variant Surface Glycoprotein (VSG) coat for survival in the mammalian bloodstream. High VSG expression within an expression site body (ESB) is mediated by RNA polymerase I (Pol I), which in other eukaryotes exclusively transcribes ribosomal RNA genes (rDNA). As T. brucei is reliant on Pol I for VSG transcription, we investigated Pol I transcription inhibitors for selective anti-trypanosomal activity. The Pol I inhibitors quarfloxin (CX-3543), CX-5461, and BMH-21 are currently under investigation for treating cancer, as rapidly dividing cancer cells are particularly dependent on high levels of Pol I transcription compared with nontransformed cells. In T. brucei all three Pol I inhibitors have IC50 concentrations for cell proliferation in the nanomolar range: quarfloxin (155 nM), CX-5461 (279 nM) or BMH-21 (134 nM) compared with IC50 concentrations in the MCF10A human breast epithelial cell line (4.44 μM, 6.89 μM or 460 nM, respectively). T. brucei was therefore 29-fold more sensitive to quarfloxin, 25-fold more sensitive to CX-5461 and 3.4-fold more sensitive to BMH-21. Cell death in T. brucei was due to rapid inhibition of Pol I transcription, as within 15 minutes treatment with the inhibitors rRNA precursor transcript was reduced 97-98% and VSG precursor transcript 91-94%. Incubation with Pol I transcription inhibitors also resulted in disintegration of the ESB as well as the nucleolus subnuclear structures, within one hour. Rapid ESB loss following the block in Pol I transcription argues that the ESB is a Pol I transcription nucleated structure, similar to the nucleolus. In addition to providing insight into Pol I transcription and ES control, Pol I transcription inhibitors potentially also provide new approaches to treat trypanosomiasis., Author summary Trypanosoma brucei is protected by an essential Variant Surface Glycoprotein (VSG) coat in the mammalian bloodstream. The active VSG gene is transcribed by RNA polymerase I (Pol I), which typically only transcribes rDNA. Pol I transcription inhibitors are under clinical trials for cancer chemotherapy. As T. brucei relies on Pol I for VSG transcription, we investigated its susceptibility to these drugs. We show that quarfloxin (CX-3543), CX-5461, and BMH-21 are effective against T. brucei at nanomolar concentrations. T. brucei death was due to rapid and specific inhibition of Pol I transcription. Incubation with Pol I transcription inhibitors also resulted in disappearance of Pol I subnuclear structures like the nucleolus and the VSG expression site body (ESB). Rapid ESB loss followed the Pol I transcription block, arguing that the ESB is nucleated by Pol I transcription. Pol I transcription inhibitors could therefore potentially function as novel drugs against trypanosomiasis.

Background: Multiple myeloma (MM) requires combination drug therapies to delay acquired drug resistance and clinical relapse. We co-developed CX-5461, a highly-selective inhibitor of RNA polymerase I-mediated rDNA transcription(1), currently in phase I trials for relapsed haematological malignancies (Peter Mac). CX-5461 produces a targeted nucleolar DNA damage response (DDR), triggering both a p53-dependent and -independent nucleolar stress response and killing malignant cells while sparing normal cells(2,3). Single-agent CX-5461 provides an impressive survival benefit in mouse models of B-cell lymphoma, acute myeloid leukaemia and now MM(2,4,5). However, drug resistance eventually occurs, confirming the need for combination therapies. Aim: To test the efficacy of CX-5461 in combination with the histone deacetylase inhibitor panobinostat, (prioritised from a boutique high-throughput screen of anti-myeloma agents), with a focus on the setting of resistance to proteasome-inhibitors (PIs). Methods: We assessed the impact of CX-5461 and panobinostat on overall survival in mouse models of MM, then surveyed the effects on cellular response and molecular markers of DDR. We developed bortezomib-resistant cell lines and an in vivo model of bortezomib-resistance to test this combination in the setting of PI-resistance. Results: CX-5461 in combination with panobinostat provides a significant survival advantage in both the transplanted Vk*MYC and the 5T33/KaLwRij models, with minimal bone marrow toxicity. The combination showed increased anti-proliferative effects and cell death in vitro. Interestingly, experiments interrogating the downstream cellular response of this combination suggest that the mechanism(s) driving synergy are complex and cell context-dependent. Cell cycle analysis indicates that both CX-5461- and panobinostat-driven cell cycle effects, i.e. G2/M and G1/S arrest, respectively, are dominant in the combination setting in a cell line-dependent manner, suggesting that context-dependent factors such as p53 may influence the cellular response. Mechanistically, in both p53-wild type and -null cell lines we observe an increase in DDR signalling with single agent CX-5461, with only moderate further increase with the combination. Moreover, CX-5461-mediated MYC downregulation is not universally observed, with the combination promoting further downregulation only in some cell lines. Given the potential for affecting global transcription programs downstream of panobinostat, we are performing transcriptome analyses in the combination setting compared to single agent treatment. We have generated bortezomib-resistant cell lines, sequentially increasing drug exposure to establish populations growing at concentrations above the IC90 of the parental lines. The resistant 5T33 cells retain their resistance to bortezomib in vivo and we have demonstrated that CX-5461 remains effective in this model, significantly increasing survival. We are currently examining the combination of CX-5461 with panobinostat in this model of bortezomib-resistance, which will give critical information guiding patient selection for future clinical trials. Conclusion: The rDNA transcription inhibitor CX-5461 synergises in vitro and in vivo with panobinostat, and CX-5461 retains efficacy in the setting of bortezomib-resistant myeloma. References Drygin et al., Cancer Research 2011 Bywater et al., Cancer Cell 2012 Quin et al, Oncotarget, 2016 Devlin et al., Cancer Discovery 2016 Hein et al., Blood 2017 Disclosures Harrison: Janssen-Cilag: Other: Scientific advisory board.

Despite the development of novel drugs, the prospects for many patients with acute myeloid leukemia (AML) remain dismal. This study reveals that the selective inhibitor of RNA polymerase I (Pol I) transcription, CX-5461, effectively treats aggressive AML, including mixed-lineage leukemia-driven AML, and outperforms standard chemotherapies. In addition to the previously characterized mechanism of action of CX-5461 (ie, the induction of p53-dependent apoptotic cell death), the inhibition of Pol I transcription also demonstrates potent efficacy in p53null AML in vivo. This significant survival advantage in both p53WT and p53null leukemic mice treated with CX-5461 is associated with activation of the checkpoint kinases 1/2, an aberrant G2/M cell-cycle progression and induction of myeloid differentiation of the leukemic blasts. The ability to target the leukemic-initiating cell population is thought to be essential for lasting therapeutic benefit. Most strikingly, the acute inhibition of Pol I transcription reduces both the leukemic granulocyte-macrophage progenitor and leukemia-initiating cell (LIC) populations, and suppresses their clonogenic capacity. This suggests that dysregulated Pol I transcription is essential for the maintenance of their leukemia-initiating potential. Together, these findings demonstrate the therapeutic utility of this new class of inhibitors to treat highly aggressive AML by targeting LICs. Refereed/Peer-reviewed

Increased rates of ribosome biogenesis and biomass accumulation are fundamental properties of rapidly growing and dividing malignant cells. The MYC oncoprotein drives growth predominantly via its ability to upregulate the ribosome biogenesis program, in particular stimulating the activity of the RNA Polymerase I (Pol I) machinery to increase ribosomal RNA (rRNA) transcription. Although MYC function is known to be highly dependent on the cellular signalling context, the pathways interacting with MYC to regulate transcription of ribosomal genes (rDNA) in vivo in response to growth factor status, nutrient availability and cellular stress are only beginning to be understood. To determine factors critical to MYC-dependent stimulation of rDNA transcription in vivo, we performed a transient expression screen for known oncogenic signalling pathways in Drosophila. Strikingly, from the broad range of pathways tested, we found that ribosomal protein S6 Kinase (S6K) activity, downstream of the TOR pathway, was the only factor rate-limiting for the rapid induction of rDNA transcription due to transiently increased MYC. Further, we demonstrated that one of the mechanism(s) by which MYC and S6K cooperate is through coordinate activation of the essential Pol I transcription initiation factor TIF-1A (RRN 3). As Pol I targeted therapy is now in phase 1 clinical trials in patients with haematological malignancies, including those driven by MYC, these data suggest that therapies dually targeting Pol I transcription and S6K activity may be effective in treating MYC-driven tumours.

Nucleotide excision DNA repair (NER) pathway mutations cause neurodegenerative and progeroid disorders (xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD)), which are inexplicably associated with (XP) or without (CS/TTD) cancer. Moreover, cancer progression occurs in certain patients, but not others, with similar C-terminal mutations in the XPB helicase subunit of transcription and NER factor TFIIH. Mechanisms driving overproliferation and, therefore, cancer associated with XPB mutations are currently unknown. Here using Drosophila models, we provide evidence that C-terminally truncated Hay/XPB alleles enhance overgrowth dependent on reduced abundance of RNA recognition motif protein Hfp/FIR, which transcriptionally represses the MYC oncogene homologue, dMYC. The data demonstrate that dMYC repression and dMYC-dependent overgrowth in the Hfp hypomorph is further impaired in the C-terminal Hay/XPB mutant background. Thus, we predict defective transcriptional repression of MYC by the Hfp orthologue, FIR, might provide one mechanism for cancer progression in XP/CS.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.

Recent findings by our group have been instrumental in the development of the novel selective inhibitors of RNA Polymerase I (Pol I) (Drygin et al., Cancer Research, 2011; Bywater et al. Cancer Cell, 2012). This work has led to the fundamental discovery that ribosomal gene transcription by Pol I is not simply a “housekeeping” process in cancer cells but is highly regulated to maintain their viability (Bywater et al. Nature Reviews Cancer, 2013). Strikingly, inhibition of Pol I transcription shows a profound selectivity for malignant over normal cells in preclinical studies. As with the majority of targeted therapies, despite initial favorable responses to approaches that target ribosome synthesis and/or function in MYC-driven lymphoma models, resistant disease emerges. It is increasingly clear that maximizing the inhibition of key signaling networks as a whole improves anti-tumor response. The well-established reliance of MYC-driven malignancies on elevated rates of ribosome biogenesis, mTORC1/eIF4E-driven protein synthesis, and cell growth makes them vulnerable to therapeutic strategies that target the ribosome. Thus we hypothesized that the simultaneous targeting of the ribosome at multiple points would antagonize the development of acquired resistance and consequently prolong survival in MYC-driven cancer models. We will present data to demonstrate that targeting both ribosome synthesis and function through the combination of novel inhibitors of RNA polymerase I transcription, and PI3K/AKT/mTOR signaling inhibitors or PIM Kinase inhibitors provides a significant increase in survival compared to treatment with single agents (Devlin et al., Cancer Discovery 2016; Rebello et al., Clinical Cancer Res. 2016). We will also discuss the molecular mechanism by which multipoint targeting of the ribosome synergizes to increase survival. Finally we will discuss our collaboration with Pimera, Inc. to develop highly selective second generation RNA Pol I inhibitors. The lead compound PMR-116 is showing exceptional activity in transgenic models of malignancy, including MLL-ENL AML and Vk*MYC driven multiple myeloma. We anticipate this compound will enter the clinic in 2017. Citation Format: Ross D. Hannan, Nadine Hein, Katherine M. Hannan, Gretchen Poortinga, Elaine Sanij, Jirawas Sornkom, Kylee MacLachlan, Andrew Cuddihy, Carleen Cullinane, Luc Furic, Denis Drygin, Mustapha Haddach, Simon Harrison, Grant McArthur, Richard B. Pearson. Drugging the ribosome at the level of synthesis and translation to treat solid and hematologic cancers. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr IA10.

hairy is a Drosophila pair-rule segmentation gene that functions genetically as a repressor. To isolate protein components of Hairy-mediated repression, we used a yeast interaction screen and identified a Hairy-interacting protein, the Drosophila homolog of the human C-terminal-binding protein (CtBP). Human CtBP is a cellular phosphoprotein that interacts with the C-terminus of the adenovirus E1a oncoprotein and functions as a tumor suppressor. dCtBP also interacts with E1a in a directed yeast two-hybrid assay. We show that dCtBP interacts specifically and directly with a small, previously uncharacterized C-terminal region of Hairy. dCtBP activity appears to be specific to Hairy of the Hairy/Enhancer of split [E(spl)]/Dpn basic helix-loop-helix protein class. We identified a P-element insertion within the dCtBP transcription unit that fails to complement alleles of a known locus, l(3)87De. We demonstrate that dCtBP is essential for proper embryonic segmentation by analyzing embryos lacking maternal dCtBP activity. While Hairy is probably not the only segmentation gene interacting with dCtBP, we show dose-sensitive genetic interactions between dCtBP and hairy mutations.

Neural fate specification in Drosophila is promoted by the products of the proneural genes, such as those of the achaete–scute complex , and antagonized by the products of the Enhancer of split [E(spl)] complex , hairy, and extramacrochaetae . As all these proteins bear a helix-loop-helix (HLH) dimerization domain, we investigated their potential pairwise interactions using the yeast two-hybrid system. The fidelity of the system was established by its ability to closely reproduce the already documented interactions among Da, Ac, Sc, and Extramacrochaetae. We show that the seven E(spl) basic HLH proteins can form homo- and heterodimers inter-se with distinct preferences. We further show that a subset of E(spl) proteins can heterodimerize with Da, another subset can heterodimerize with proneural proteins, and yet another with both, indicating specialization within the E(spl) family. Hairy displays no interactions with any of the HLH proteins tested. It does interact with the non-HLH protein Groucho, which itself interacts with all E(spl) basic HLH proteins, but with none of the proneural proteins or Da. We investigated the structural requirements for some of these interactions by site-specific and deletion mutagenesis.

MYC deregulation is common in human cancer. IG-MYC translocations that are modeled in Eμ-Myc mice occur in almost all cases of Burkitt lymphoma as well as in other B-cell lymphoproliferative disorders. Deregulated expression of MYC results in increased mTOR complex 1 (mTORC1) signaling. As tumors with mTORC1 activation are sensitive to mTORC1 inhibition, we used everolimus, a potent and specific mTORC1 inhibitor, to test the requirement for mTORC1 in the initiation and maintenance of Eμ-Myc lymphoma. Everolimus selectively cleared premalignant B cells from the bone marrow and spleen, restored a normal pattern of B-cell differentiation, and strongly protected against lymphoma development. Established Eμ-Myc lymphoma also regressed after everolimus therapy. Therapeutic response correlated with a cellular senescence phenotype and induction of p53 activity. Therefore, mTORC1-dependent evasion of senescence is critical for cellular transformation and tumor maintenance by MYC in B lymphocytes. Significance: This work provides novel insights into the requirements for MYC-induced oncogenesis by showing that mTORC1 activity is necessary to bypass senescence during transformation of B lymphocytes. Furthermore, tumor eradication through senescence elicited by targeted inhibition of mTORC1 identifies a previously uncharacterized mechanism responsible for significant anticancer activity of rapamycin analogues and serves as proof-of-concept that senescence can be harnessed for therapeutic benefit. Cancer Discov; 3(1); 82–95. ©2012 AACR. This article is highlighted in the In This Issue feature, p. 1

SummaryIncreased transcription of ribosomal RNA genes (rDNA) by RNA Polymerase I is a common feature of human cancer, but whether it is required for the malignant phenotype remains unclear. We show that rDNA transcription can be therapeutically targeted with the small molecule CX-5461 to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population. The therapeutic effect is a consequence of nucleolar disruption and activation of p53-dependent apoptotic signaling. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. These results identify selective inhibition of rDNA transcription as a therapeutic strategy for the cancer specific activation of p53 and treatment of hematologic malignancies.

Loss of c-MYC is required for downregulation of ribosomal RNA (rRNA) gene (rDNA) transcription by RNA Polymerase I (Pol I) during granulocyte differentiation. Here, we demonstrate a robust reduction of Pol I loading onto rDNA that along with a depletion of the MYC target gene upstream binding factor (UBF) and a switch from epigenetically active to silent rDNA accompanies this MYC reduction. We hypothesized that MYC may coordinate these mechanisms via direct regulation of multiple components of the Pol I transcription apparatus. Using gene expression arrays we identified a 'regulon' of Pol I factors that are both downregulated during differentiation and reinduced in differentiated granulocytes upon activation of the MYC-ER transgene. This regulon includes the novel c-MYC target genes RRN3 and POLR1B. Although enforced MYC expression during granulocyte differentiation was sufficient to increase the number of active rRNA genes, its activation in terminally differentiated cells did not alter the active to inactive gene ratio despite increased rDNA transcription. Thus, c-MYC dynamically controls rDNA transcription during granulocytic differentiation through the orchestrated transcriptional regulation of core Pol I factors and epigenetic modulation of number of active rRNA genes. ispartof: Nucleic Acids Research vol:39 issue:8 pages:3267-81 ispartof: location:England status: published

In mammals, the mechanisms regulating the number of active copies of the ∼200 ribosomal RNA (rRNA) genes transcribed by RNA polymerase I are unclear. We demonstrate that depletion of the transcription factor upstream binding factor (UBF) leads to the stable and reversible methylation-independent silencing of rRNA genes by promoting histone H1–induced assembly of transcriptionally inactive chromatin. Chromatin remodeling is abrogated by the mutation of an extracellular signal-regulated kinase site within the high mobility group box 1 domain of UBF1, which is required for its ability to bend and loop DNA in vitro. Surprisingly, rRNA gene silencing does not reduce net rRNA synthesis as transcription from remaining active genes is increased. We also show that the active rRNA gene pool is not static but decreases during differentiation, correlating with diminished UBF expression. Thus, UBF1 levels regulate active rRNA gene chromatin during growth and differentiation.

c-MYC inhibits differentiation and regulates the process by which cells acquire biomass, cell growth. Down-regulation of c-MYC, reduced cell growth, and decreased activity of the PI3K/AKT/mTORC1 signal transduction pathway are features of the terminal differentiation of committed myeloid precursors to polymorphonuclear neutrophils. Since mTORC1 regulates growth, we hypothesized that pharmacological inhibition of mTORC1 by rapamycin may reverse the phenotypic effects of c-MYC. Here we show that granulocytes blocked in their ability to differentiate by enforced expression of c-MYC can be induced to differentiate by reducing exogenous c-MYC expression through rapamycin treatment. Rapamycin also reduced expression of endogenous c-MYC and resulted in enhanced retinoid-induced differentiation. Total cellular c-Myc mRNA and c-MYC protein stability were unchanged by rapamycin, however the amount of c-Myc mRNA associated with polysomes was reduced. Therefore rapamycin limited expression of c-MYC by inhibiting c-Myc mRNA translation. These findings suggest that mTORC1 could be targeted to promote terminal differentiation in myeloid malignancies characterized by dysregulated expression of c-MYC.

The MYC oncoprotein and transcription factor has a well-described role in harnessing and driving ribosome biogenesis (RiBi) via its regulation of all three RNA polymerases, and it is proposed that this is a crucial output of MYC's malignant function. Thus, the potential for therapeutic intervention in a broad range of MYC-driven cancers may converge on targeting the RiBi program, potentially as monotherapy or, more likely, as a basis for rational combination therapies. Indeed, small molecule (CX-5461) inhibition of RNA Polymerase I (Pol I), a major MYC target, selectively kills MYC-driven lymphoma in vivo via activation of a p53-dependent nucleolar stress response. While CX-5461 potently inhibits ribosome biogenesis in a MYC-driven B-cell lymphoma via p53-dependent apoptosis, it is unclear whether all malignancies will respond similarly. Furthermore, as we predict resistance to CX-5461 and tumor relapse from our in vivo mouse studies, the need to identify pathways responsible for mediating resistance to Pol I inhibition in the background of oncogenic MYC is necessary for future development of rational combination therapies. To address these key questions, we have undertaken multiple approaches. Firstly, we are utilizing model systems of acute myeloid leukemia (AML) to take both an unbiased and a direct approach to understanding the role of MYC in mediating CX-5461 sensitivity. Secondly, to identify factors conferring acquired resistance to CX-5461 we have used the well-characterized Eµ-Myc mouse model of MYC-mediated lymphoma where, following a period of disease remission, prolonged dosing of CX-5461 in mice bearing lymphomas results in eventual relapse due to acquired drug resistance. Thus, we compared whole-exome sequencing data between isogenic sensitive and CX-5461 resistant B-cell lymphomas to identify additional mutations conferring drug resistance. Finally, we are using the Eµ-Myc model to investigate the regulation of rDNA transcription itself during cancer progression. Compared to wild type B-cells, malignant MYC over-expressing B-cells have hyper-activated Pol I and increased rDNA transcription. This increase in transcription is associated with a dramatic increase in the number of transcriptionally active rDNA repeats, indicating changes in chromatin structure during transformation. In order to assess epigenetic changes at the rDNA loci and to identify long distance rDNA interactions occurring during malignant progression, we have used ChIP-seq and circularized chromosome conformation capture sequencing (4C-seq) at all cell stages of malignancy in vivo (wild type, pre-malignant and malignant Eµ-Myc B-cells). We demonstrate that transition from premalignancy to malignancy is associated with robust changes in rDNA chromatin as well as reorganization of rDNA-genome interactions with a significant increase in these interactions being detected in malignant cells. Our data provide evidence for MYC-driven activation of rDNA in cancer progression beyond ribosome biogenesis and offer novel insights into the spatial and transcriptional dynamics of the rDNA-associated genome during malignant transformation. We will present our latest data on all fronts aimed at understanding the critical role that MYC plays in therapeutic inhibition of Pol I transcription. Citation Format: Gretchen Poortinga, Jeannine Diesch, Elaine Sanij, Nadine Hein, Jirawas Sornkum, Donald Cameron, Megan J. Bywater, Ricky Johnstone, Denis Drygin, Sean O'Brien, Richard B. Pearson, Grant A. McArthur, Ross D. Hannan. Elucidating mechanisms that cooperate with the therapeutic inhibition of RNA polymerase I to treat MYC-driven hematological malignancies. [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 A31.

The regulation of cell mass (cell growth) is often tightly coupled to the cell division cycle (cell proliferation). Ribosome biogenesis and the control of rDNA transcription through RNA polymerase I are known to be critical determinants of cell growth. Here we show that granulocytic cells deficient in the c-MYC antagonist MAD1 display increased cell volume, rDNA transcription and protein synthesis. MAD1 repressed and c-MYC activated rDNA transcription in nuclear run-on assays. Repression of rDNA transcription by MAD1 was associated with its ability to interact directly with the promoter of upstream binding factor (UBF), an rDNA regulatory factor. Conversely, c-MYC activated transcription from the UBF promoter. Using siRNA, UBF was shown to be required for c-MYC-induced rDNA transcription. These data demonstrate that MAD1 and c-MYC reciprocally regulate rDNA transcription, providing a mechanism for coordination of ribosome biogenesis and cell growth under conditions of sustained growth inhibition such as granulocyte differentiation.

Genetically-engineered mouse models offer controlled and reproducible systems in which to study the genetics of cancer development. We have used massively-parallel sequencing to provide a comprehensive analysis of the step-wise progression of cancer in the highly tractable Eμ-Myc mouse model. The Eμ-Myc mouse has been engineered to harbor a t(8;14) translocation juxtaposing the IgH enhancer to the proto-oncogene Myc, a lesion frequently observed in mature B-cell malignancies. Eμ-Myc mice develop spontaneous Burkitt-like B-cell leukemia/lymphoma with 100% penetrance through predicable premalignant to malignant progression phases. Malignant progression ostensibly occurs as a result of secondary somatic mutations that co-operate with Myc. Despite common perception that the majority of tumors can be explained by disruption of the archetypal Cdkn2a and Trp53 tumor suppressor axis, in more than half of spontaneous Eμ-Myc tumors harbor secondary mutations that remain unknown. We hypothesize that the identification of genes that cooperate with Myc could therefore yield not only new biological insight into oncogenic pathways involved in B-cell lymphomagenesis but also improve therapy for patients in the future. We applied exome-sequencing to a cohort of 18 spontaneous murine Eμ-Myc lymphomas to determine novel somatic driver mutations capable of cooperating with Myc. In addition to exome-sequencing, we harvested blood samples at 2-week intervals throughout the premalignant to malignant phases to allow a temporal analysis of mutations that were detected at end-stage. End-stage tumors were found to be heterogeneous in nature, ranging from 11 to 36 single-base mutations, deletions or insertions. The screen identified genes previously described to be mutated in Eμ-Myc lymphoma (Trp53, Cdkn2a, Kras and Nras). Interestingly, concurrent mutations in known cancer genes were evident in clonal tumors from the sequenced cohort with, for example, a Cdkn2a deletion co-occurring with an activating Kras mutation. In addition to these mutations, we identified novel protein-truncating mutations in Bcl6 co-repressor (Bcor), which has not previously been described in Eμ-Myc lymphoma. Bcor truncating mutations were present at a high frequency, occurring in 3 out of 18 samples. RNAi mediated knockdown of Bcor in Eμ-Myc fetal liver hematopoietic stem cells reconstituted into lethally irradiated congenic recipient mice accelerated lymphomagenesis, validating Bcor as a tumor suppressor gene in this model. BCOR loss-of-function mutations have recently been identified in human cancers suggesting that BCOR acts as a tumor-suppressor in a wide range of malignancies. Given that Bcor mutations frequently occur in the Eμ-Myc lymphoma, we hypothesize that Bcor is critically important for B-cell development and that its disruption can induce malignancy in co-operation with other potent oncogenes such as Myc. Citation Format: Marcus P. Lefebure, Richard Tothill, Jason Li, Geoff Matthews, Jake Shortt, Edwin Hawkins, Elizabeth Kruse, Maria Doyle, Gretchen Poortinga, Ross Hannan, Vivian Bardwell, Micah Gearhart, Ricky W. Johnstone. Exome sequencing reveals Bcl6 co-repressor (Bcor) as a frequently mutated tumor suppressor gene in Eμ-Myc lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 449. doi:10.1158/1538-7445.AM2014-449

We recently demonstrated that transcription of the ribosomal genes (rRNA) by RNA Polymerase I (Pol I) can be therapeutically targeted with a novel small molecule, CX-5461, to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B-cell population (Bywater et al Cancer Cell 2012; Bywater et al Nature Reviews Cancer 2013). The therapeutic effect was a consequence of nucleolar disruption, activation of ribosomal protein (Rp)-MDM2-P53 nucleolar stress response and apoptosis. We have recently launched a first-in-human clinical trial of CX-5461 in patients with hematological malignancies and although our pre-clinical data indicate immense potential of Pol I targeting for cancer therapy, some cancers still develop resistance. We hypothesized that simultaneously targeting the ribosome at multiple steps will extend survival. Thus we tested pharmacological inhibitors of PI3K/AKT/mTOR signalling in combination with CX-5461 as the former pathway is known to potently regulate both translational activity (Jefferies et al EMBO J, 1997; Pourdehnad M et al PNAS 2013) and ribosome biogenesis (Chan et al Science Signaling 2011; Devlin et al FEBS J 2013; Wall et al Cancer Discovery 2013). Using the Eμ-Myc model of B-cell lymphoma we demonstrate that multiple pharmacological inhibitors of the PI3K/AKT/mTOR pathway suppress transcription of the rRNA genes and induce cell death similar to CX-5461. Unexpectedly however, PI3K/AKT/mTOR pathway blockade is not associated with nucleolar disruption, or activation of the Rp/MDM2/p53 nucleolar stress pathway. This is because inhibition of PI3K/AKT/mTOR signalling suppresses both rRNA synthesis and ribosomal protein synthesis equally and therefore does not increase the pool of free Rps necessary to suppress MDM2 E3 ligase that regulates p53 stability. Furthermore, we demonstrate that combined treatment of Eμ-Myc tumor-bearing mice with CX-5461 and Everolimus delayed relapse compare to single agent and significantly extended survival of tumor bearing mice. These data demonstrate that dual targeting of the ribosome by selectively inhibiting Pol I transcription and inhibition of key signaling molecules regulating ribosome synthesis and function combine to potently treat MYC driven tumors. It therefore provides a rationale to combine such drugs in the clinic for the treatment of MYC driven cancer. Citation Format: Richard B. Pearson, Jennifer R. Devlin, Katherine M. Hannan, Nadine Hein, Megan J. Bywater, Gretchen Poortinga, Donald Cameron, Denis Drygin, Sean O'Brien, Carleen Cullinane, Grant A. McArthur, Ross D. Hannan. Multi-point targeting of the synthetic lethal interactions between Myc, ribosome biogenesis and ribosome function cooperates to treat B-cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2735. doi:10.1158/1538-7445.AM2014-2735

Introduction: We recently demonstrated that transcription of the ribosomal genes (rDNA) by RNA Polymerase I (Pol I) can be therapeutically targeted with a novel small molecule, CX-5461, to selectively kill B-lymphoma cells in vivo while maintaining a viable wild-type B cell population (Bywater et al Cancer Cell 2012; Bywater et al Nature Reviews Cancer 2013). The therapeutic effect was a consequence of nucleolar disruption and activation of ribosomal protein (Rp)-MDM2-P53 nucleolar stress response (3) leading to apoptosis. Human leukemia and lymphoma cell lines also show high sensitivity to inhibition of rDNA transcription that is dependent on p53 mutational status. As our pre-clinical data indicate immense potential of Pol I targeting as a cancer therapy, we have launched a first-in-human clinical trial of CX-5461 in patients with hematological malignancies. Despite impressive initial responses with CX-5461 as a single agent, we decided to explore its combination potential. We hypothesised that simultaneously targeting the ribosome at multiple steps would reduce the instance of acquired resistance to CX-5461 and extend survival. Thus we tested pharmacological inhibitors of the PI3K/AKT/mTOR pathway in combination with CX-5461 as the former signaling molecules are known to be potent regulators of both ribosome translational activity (Jefferies et al EMBO J, 1997; Pourdehnad M et al PNAS 2013) and ribosome biogenesis (Chan et al Science Signaling 2011; Devlin et al FEBS J 2013). Experimental procedures and new data: Using the Eμ-Myc model of B-cell lymphoma we demonstrate that multiple pharmacological inhibitors of the PI3K/AKT/mTOR pathway also suppress transcription of the rRNA genes and induce cell death to a similar extent as CX-5461. Unexpectedly however, PI3K/AKT/mTOR pathway blockade is not associated with nucleolar disruption, nor activation of the Rp/MDM2/p53 nucleolar stress pathway. We demonstrate this is because inhibition of PI3K/AKT/mTOR suppresses both rRNA synthesis and ribosomal protein synthesis equally and therefore does not result in a pool of free Rps that are necessary to suppress MDM2 E3 ligase that regulates p53 stability. Instead apoptosis induced by PI3K/AKT/mTOR was associated with up-regulated expression of the pro-apoptotic BH3-only protein BMF. Furthermore, we demonstrate that combined treatment of Eμ-Myc tumour-bearing mice with CX-5461 and Everolimus delayed relapse compare to single agent and significantly extended survival of tumor bearing mice. Conclusions: These data demonstrate that dual targeting of the ribosome by selectively inhibiting Pol I transcription and by inhibition of key signaling molecules regulating ribosome synthesis and function combine to potently treat MYC driven tumours and provides a rationale to combine such drugs in the clinic for the treatment of MYC driven cancer. Moreover these data also demonstrate that MYCs control of Pol I transcription and nucleolar integrity is required for its oncogenic potential, independent of its well described function in function in controlling ribosome levels and protein translation. This abstract is also presented as poster C64. Citation Format: Ross Hannan, Jennifer Devlin, Katherine Hannan, Nadine Hein, Megan Bywater, Gretchen Poortinga, Don Cameron, Denis Drygin, Carleen Cullinane, Sean O'Brien, Grant McArthur, Richard Pearson. Combined inhibition of ribosome function and ribosomal RNA gene transcription cooperate to delay relapse and extend survival in MYC-driven tumors. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr PR16.