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Background: The role of dysregulation of the proto-oncogene MYC in both early and late myeloma progression events is well established. Among key MYC -downstream targets is upregulation of ribosomal biogenesis, resulting in increased protein translational capacity and biomass accumulation that is characteristic of neoplastic cells. Thus, given the relationship between myeloma pathobiology, MYC dysregulation, and ribosomal biogenesis, we hypothesized that selective targeting of ribosomal RNA (rRNA) transcription with the small molecule RNA polymerase (pol) I inhibitor CX-5461 (Senhwa Biosciences) may represent a novel therapeutic strategy in myeloma. Methods: Studies with CX-5461 were performed in human myeloma cell lines, isogenic p53 wild-type (wt) and knock-out (KO) p53 cells generated using sequence-specific zinc-finger nucleases, drug-resistant cell lines, primary patient samples, and myeloma murine xenograft models using NOD-SCID IL2Rgnull mice. Results: CX-5461 treatment of p53 wt (MM1.S, MOLP-8) and p53 mutant (U266, RPMI-8226) myeloma cell lines demonstrated a time- and dose-dependent decrease in cell proliferation with a median inhibitory concentration (IC50) at nM levels after 72 hours. A corresponding increase in cleaved-PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot as well as increased Annexin V staining on flow cytometry analysis, although this was more pronounced in p53 wt versus mutant cell lines. CX-5461 also retained activity in a panel of cell lines resistant to standard myeloma therapeutic agents (bortezomib, carfilzomib, lenalidomide, and doxorubicin) and in primary patient samples, including a heavily pretreated relapsed/refractory patient and a de novo plasma cell leukemia patient with del 17p. In vivo studies using a systemic isogenic MM1.S p53 wt and KO myeloma murine xenograft model demonstrated significant improvement in median overall survival in the CX-5461-treated p53 wt cohort (41 days vs. not reached, P .05), although outcomes were more modest in the p53 KO cohort with only a trend towards improved survival (P.1) in the drug-treated mice. To probe the p53-independent effects of CX-5461, gene expression profiling and gene set enrichment analysis was performed on isogenic MM1.S and MOLP-8 p53 wt and KO myeloma cell lines treated with CX-5461 or vehicle. These results suggested downregulation of MYC downstream targets as one p53-independent effect of RNA pol I inhibition. qPCR and Western blot studies revealed rapid downregulation of MYC at the transcript level within 1-hour of CX-5461 treatment followed by decreases in MYC protein levels. Previous studies have suggested ribosomal biogenesis is tightly controlled by an auto-regulatory feedback mechanism in which ribosomal proteins such as RPL5 and RPL11 can bind to the 3'UTR of MYC mRNA and facilitate its degradation through the RNA-induced silencing complex (RISC). Because RNA pol I inhibition is known to induce a nucleolar stress response and increase the availability of free ribosomal proteins, RISC-mediated degradation of MYC mRNA was explored as one possible mechanism of CX-5461-mediated MYC downregulation. Indeed, treatment with CX-5461 led to increased pull-down of RPL5 when immunoprecipitated with the RISC subunit TAR (HIV-1) RNA Binding Protein 2 (TARBP2) compared to vehicle-treated controls, and RNA immunoprecipitation assays with the catalytic RISC subunit, Argonaute 2 (AGO2), demonstrated enrichment of MYC mRNA with CX-5461 treatment. These results suggest that CX-5461 may induce degradation of MYC through the cooperative binding of ribosomal proteins, RISC subunits, and MYC mRNA. Finally, to evaluate the role of MYC expression and ribosomal biogenesis in relation to CX-5461 sensitivity, MYC was overexpressed in the H1112 myeloma cell line, which at baseline does not harbor a MYC translocation. MYC overexpression in H1112pCDH-myc cells led to increased basal pre-rRNA transcript levels compared to H1112pCDH cells, and furthermore, led to enhanced sensitivity to CX-5461. Conclusion: RNA pol I inhibition by CX-5461 is a promising target in myeloma therapy, with downregulation of MYC representing one mechanism of action. Moreover, increased MYC expression enhances sensitivity to CX-5461, providing rationale for the clinical translation of CX-5461 for the treatment of myeloma and other MYC-driven cancers. Disclosures O'Brien: Senhwa Biosciences, Inc.: Employment. Keats:Translational Genomic Research Institute: Employment. Orlowski:Bristol-Myers Squibb: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Spectrum Pharmaceuticals: Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Acetylon: Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Research Funding; Millennium Pharmaceuticals: Consultancy, Research Funding; Forma Therapeutics: Consultancy; Genentech: Consultancy; BioTheryX, Inc.: Membership on an entity's Board of Directors or advisory committees; Array BioPharma: Consultancy, Research Funding.

Malignant transformation is commonly associated with dysregulated ribosome biogenesis and for more than 100 years, pathologists have utilized the increase in size and number of nucleoli, the site of ribosome biogenesis, as a marker for aggressive malignancies (Hein et al., Trends in Molecular Medicine, 2013). Recently we demonstrated that hyperactivated RNA Polymerase I (Pol I) transcription, a rate limiting step in ribosome biogenesis, can be specifically targeted by the small molecule inhibitor, CX-5461 (Drygin et al., Cancer Research, 2011; Bywater et al. Cancer Cell, 2012). When evaluated for its anti-proliferative activity against a panel of genetically diverse human cancer cell lines, those derived from p53 wild type hematological malignancies were the most sensitive (Drygin et al., Cancer Research, 2011). Evaluation of whether Pol I transcription inhibition could serve as therapeutic strategy in vivo revealed that CX-5461 administration significantly prolongs the overall survival of Em-Myc lymphoma bearing mice. This survival advantage was associated with rapid activation of the ribosomal protein (Rp)-MDM2-p53 nucleolar stress response (Deisenroth et al., Oncogene, 2010) and subsequent induction of tumour cell-specific p53-dependent apoptosis; importantly the wild type B-cell population was maintained and did not activate p53 pathways (Bywater et al. Cancer Cell, 2012). To explore the therapeutic potential of Pol I transcription inhibition via CX-5461 in hematological malignancies refractory to standard chemotherapy, we employed mouse models of highly aggressive MLL-driven Acute Myeloid Leukemia (AML) (MLL/AF9 + Nras and MLL/ENL + Nras) (Zuber et al., Genes Dev, 2009) and V*κ-Myc-driven Multiple Myeloma (MM) (Chesi et al. Cancer Cell, 2009). CX-5461 administration significantly increased the overall survival time of AML-bearing mice (MLL/ENL Nras median 17 days for vehicle vs 36 days for drug, P In ongoing experiments with MM-bearing mice, chronic CX-5461 administration robustly reduced the secretion of serum monoclonal Ig, as detected by serum protein electrophoresis (SPEP), and significantly prolonged the overall survival time (Figure 2) (V*κ-Myc median >160 days for drug vs, 103.5 days for vehicle, ongoing) demonstrating that CX-5461 exhibits potent anti-tumour activity in MM model. Analysis of the molecular mechanism responsible for the therapeutic benefit of CX-5461 in MM is ongoing but appears to involve both p53 mediated apoptosis and aberrant cell cycle progression. Figure 2Figure 2. These data demonstrate that hyperactivated Pol I transcription can be successfully targeted through small molecule inhibitors to treat models of human AML and MM that are refractory to standard therapy. Based on these and previously published results (Bywater et al., Cancer Cell, 2012), we have recently initiated a first-in-human, phase I, dose escalation clinical trial of this first-in-class drug, CX-5461 in patients with advanced hematological malignancies. The toxicity, response, pharmacokinetic and co-relative data from this trial will offer valuable insights as to whether inhibition of ribosome synthesis might offer a new non-genotoxic therapeutic approach in the fight against cancers, which are highly refractory to standard therapies. Disclosures: O'Brien: Senhwa Biosciences, Inc: Employment.

Background Ribosomal RNA (rRNA) forms a major component of ribosomes, which play a critical role in cellular growth and proliferation through regulation of protein synthesis. Increased rRNA transcription and ribosomal biogenesis have been associated with tumorigenesis, and recent reports have suggested that inhibition of rRNA transcription by CX-5461 (Senhwa Biosciences), a novel selective small-molecule inhibitor of RNA polymerase (pol) I, induces cell death in several human tumor types by both p53-dependent and independent mechanisms. These findings led to our hypothesis that selective RNA pol I inhibition with CX-5461 could be a rational new approach to therapy for both wild-type (wt) and mutant p53 multiple myeloma models. Methods Studies with CX-5461 were performed in wt p53 and mutant p53 cell lines, zinc-finger nuclease (ZFN) p53 knock-out (KO) isogenic myeloma cell lines, and bortezomib and carfilzomib-resistant myeloma cell lines. Results Treatment of wt p53 (MM1.S, MOLP8) and mutant p53 (U266, RPMI-8226) myeloma cell lines demonstrated a time and dose dependent decrease in cell proliferation after exposure to CX-5461 with a median inhibitory concentration (IC50) range of 50-100 nM after a 72-hour incubation. A corresponding increase in cleaved PARP, cleaved caspase-9, and cleaved caspase-3 expression was seen on Western blot, as well as increased Annexin V staining on flow cytometry analysis. Notably, the degree of Annexin V staining was less in the p53 mutant cell lines compared to the wt p53 cells at any given drug concentration, but strong apoptotic signaling could be induced in mutant p53 cell lines when using higher concentrations of CX-5461. In addition, co-culturing myeloma cells with GFP+ HS5 stromal cells to mimic the bone marrow microenvironment did decrease the therapeutic effect of CX-5461, but again could be overcome with higher drug concentrations [250-500 nM]. Similar results were seen when isogenic MM1.S ZFN p53 KO cells were used, whose sensitivity to CX-5461 was comparable to that of wt p53 cells. Finally, CX-5461 was also tested on drug-resistant myeloma cell lines that were generated by exposing cells to low concentrations of bortezomib (RMPI-8266, KAS-6/1, ANBL-6) or carfilzomib (KAS-6/1) over time. These drug-resistant cell lines showed sensitivity to CX-5461 with an IC50 in the 100-250 nM concentration range. Gene expression profiling (GEP) of isogenic MM1.S ZFN p53 KO and wt cells revealed that gene expression perturbations by CX-5461 were primarily p53-independent. Additional GEP and pathway analysis in other isogenic ZFN p53 wt and KO cell lines is currently ongoing, with a particular interest in p53-independent mechanisms that may explain the efficacy of CX-5461 in both wt and mutant p53 myeloma models. Conclusion RNA pol I inhibition by CX-5461 is a promising approach to myeloma therapy, with low nanomolar drug activity seen in wt p53, mutant p53, and drug-resistant myeloma cell line models, providing a rationale for translation of CX-5461 into the clinic for the treatment of multiple myeloma. Disclosures: O'Brien: Senhwa Biosciences, Inc: Employment. Orlowski:Bristol-Myers Squibb: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Millennium: The Takeda Oncology Company: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Onyx: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding; Resverlogix: Research Funding; Array Biopharma: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Merck: Membership on an entity’s Board of Directors or advisory committees.

Abstract 787 Multiple myeloma (MM) is an incurable malignancy characterized by infiltration of malignant plasma cells in the bone marrow. There are many subtypes of MM each of which relies on distinct oncogenic pathways. Oncogene addiction has been regarded as an Achilles' heel of cancer and has consequently been the focus of extensive efforts to design molecularly targeted anticancer therapies based on the premise that silencing an oncogene's expression will prove lethal in certain cancers. However, recent evidence suggests that MM may have a fatal vulnerability that is better described as non-oncogene addiction. Non-oncogenes do not directly transform cells but rather encode for normal cellular proteins that become overexpressed or otherwise deregulated in order to maintain oncogenic signaling. Protein kinase CK2 represents the prototypical non-oncogene. CK2 activity and expression levels are elevated in many cancers and it has been reported that MM cells rely on elevated CK2 for survival. CK2 is a highly conserved, constitutively active serine/threonine protein kinase that regulates multiple pathways important in MM including PI3K/AKT, JAK-STAT and NF-κB signaling cascades. Further, CK2 is elevated under hypoxic conditions regulating hypoxia-inducible transcription factor 1 alpha (HIF-1α) activity and other mediators of angiogenesis that are known to play an important role in MM pathogenesis. Given the generalized overexpression of CK2 in cancers, the central role of CK2 in multiple pro-survival pathways and the evidence that CK2 is an essential protein for MM cell survival, the development of selective CK2 inhibitors has emerged as an attractive targeted approach for the treatment of MM. Herein we describe CX-4945, a selective, orally bioavailable, small molecule inhibitor of CK2. CX-4945 is a potent, ATP-competitive inhibitor of both isoforms of the CK2 catalytic subunits CK2α and CK2α' with IC50 values of 1 nM. In a panel of 13 MM cell lines, CX-4945 exhibited a range of antiproliferative activity in the low micromolar range as measured by CellTiter-Glo® Luminescent Cell Viability Assay. Molecular characterization of the MM cell line panel revealed that on average CK2α mRNA levels and CK2α protein levels were elevated 9.5-fold and 14-fold, respectively, compared to normal CD138+ plasma cells. CX-4945 effectively reduced CK2 enzymatic activity in the MM cell lines. In immunoblots, the attenuation of PI3K/Akt signaling by CX-4945 was evidenced by dephosphorylation of Akt on the CK2-specific Ser129 site as well as the Ser473 and Thr308 sites. Further evidence of diminished Akt activity was demonstrated by decreased phosphorylation of the downstream Akt target protein p21 at Thr145. An additional biochemical consequence of CK2 inhibition was noted as CX-4945 selectively reduced phosphorylation of the p65 subunit of NF-κB at the CK2-specific Ser529 phosphorylation site but not at the S536 site. Moreover, CX-4945 inhibited the JAK/STAT pathway as demonstrated by the reduction in phosphorylation of regulatory sites on STAT3 (Tyr705) and JAK2 (Tyr1007/1008). PCR Arrays were used to analyze the global effect of CK2 inhibition on the NFκB and JAK/STAT mediated signal transduction pathways. Several key mediators, e.g. IL-6, CyclinD1, MYC, STAT1, STAT3, IFNγ, and IL-1R, were significantly downregulated upon treatment with CX-4945. Furthermore, CX-4945 reduced IL-6 secretion in a concentration dependent manner. In culture, MM cell lines have adapted to maintain HIF1α protein expression. CX-4945 effectively inhibited HIF1α expression and a well-known HIF1α transcriptional target, VEGF. Inhibition of CK2 by CX-4945 induced apoptosis in MM cells as shown by an increase in Caspase 3/7 activity and PARP cleavage. Interestingly, CX-4945 failed to induce apoptosis and had no effect on viability in the normal human-derived stromal cell line HS-5. These data lend further support to the CK2 non-oncogene addiction hypothesis in MM. A Phase 1 clinical trial evaluating CX-4945 as a single agent in solid tumors has already established a favorable pharmacokinetic, pharmacodynamic and safety profile. These findings, coupled with our preclinical data, suggest CX-4945 is a promising therapeutic agent for targeting aberrant CK2 activity in MM and a Phase 1 clinical trial in MM is underway. Disclosures: No relevant conflicts of interest to declare.

Abstract 1726 Poster Board I-752 The PIM family of serine/threonine kinases are pro-proliferative kinases activated by multiple cytokines and growth factor signaling. The Pim kinases are unusual in that they are regulated primarily by transcription and not by membrane recruitment or phosphorylation like other serine/threonine kinases. Activated cytokine receptors recruit JAKs to induce STAT-dependent transcription of the Pim genes. They are proto-oncogenes and have been implicated in the process of lymphomagenesis and malignant transformation. Pim overexpression has been reported in diffuse B cell lymphoma, chronic lymphocytic leukemia, FLT3-mediated acute myelogenous leukemia and prostate cancer. Pim-2 is over expressed in leukemias and lymphomas, whereas Pim-3 overexpression has been observed in melanoma, pancreatic and gastric tumors. The recent reports of elevated levels of Pim-1 expression in human prostate tumor biopsies implicate the Pim family of protein kinases in the progression of human prostate tumors. Further, in transgenic animal models, Pim-1 expression has been shown to be elevated in prostate tumors that are caused by overexpression of the c-myc oncogene. Recent evidence reveals the overlapping and compensatory nature of Pim-1 and Pim-2 phosphorylation and highlights the importance of inhibiting all isoforms. The emerging role of the PIM kinase family in hematological malignancies and solid tumors and the druggable nature of their ATP binding pocket make them attractive targets for anticancer drug development Utilizing a highly distinct molecular scaffold, CX-6258 was developed as a selective and potent small molecule pan-PIM kinase inhibitor. CX-6258 inhibits Pims 1, 2 and 3 with IC50 values in the low nanomolar range and high selectivity as evidenced in a screening panel of over 100 kinases. CX-6258 demonstrates potent in vitro antiproliferative activity, particularly in leukemia derived cell lines expressing the FLT3-ITD. Moreover, CX-6258 inhibits the phosphorylation of BAD and 4EBP1, known substrates for PIMs 1, 2 and 3. When delivered orally, this pan-Pim inhibitor is well tolerated and demonstrates potent antitumor activity in murine xenograft models of PIM driven cancer. Using CX-6258 as our “path finder” molecule, we have created four additional unique chemical scaffolds as pan-Pim inhibitors, and certain of these scaffolds can inhibit Pims 1, 2 and 3 in the picomolar range while exhibiting no inhibitory activity of the Flt3 protein kinase. The in vivo and in vitro profiles of these chemically diverse series are indicative of an effective and potent anti-cancer mechanism mediated through the selective inhibition of PIM kinase activity. Together, these findings exemplify that we have created multiple proprietary chemical series of pan-Pim inhibitors exhibiting picomolar potency and high selectivity. Disclosures No relevant conflicts of interest to declare.