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New delivery vehicles and routes of delivery were developed for the capuramycin analogue SQ641. While this compound has remarkable in vitro potency against Mycobacterium tuberculosis , it has low solubility in water and poor intracellular activity. We demonstrate here that SQ641 dissolved in the water-soluble vitamin E analogue α-tocopheryl polyethylene glycol 1000 succinate (TPGS) or incorporated into TPGS-micelles has significant activity in a mouse model of tuberculosis.

Pancreas ductal adenocarcinoma (PDAC) is a highly lethal cancer that typically presents as advanced, unresectable disease. This invasive tendency, coupled with intrinsic resistance to standard therapies and genome instability, are major contributors to poor long-term survival. The genetic elements governing the invasive propensity of PDAC have not been well elucidated. Here, in the course of validating resident genes in highly recurrent and focal amplifications in PDAC, we have identified Rio Kinase 3 (RIOK3) as an amplified gene that alters cytoskeletal architecture as well as promotes pancreatic ductal cell migration and invasion. We determined that RIOK3 promotes its invasive activities through activation of the small G protein, Rac. This genomic and functional link to Rac signaling prompted a genome wide survey of other components of the Rho family network, revealing p21 Activated Kinase 4 (PAK4) as another amplified gene in PDAC tumors and cell lines. Like RIOK3, PAK4 promotes pancreas ductal cell motility and invasion. Together, the genomic and functional profiles establish the Rho family GTP-binding proteins as integral to the hallmark invasive nature of this lethal disease.

Summary Multiple myeloma (MM) evolves from a highly prevalent premalignant condition termed MGUS. The factors underlying the malignant transformation of MGUS are unknown. We report a MGUS/MM phenotype in transgenic mice with Eμ-directed expression of the XBP-1 spliced isoform (XBP-1s), a factor governing unfolded protein/ER stress response and plasma-cell development. Eμ-XBP-1s elicited elevated serum Ig and skin alterations. With age, Eμ-xbp-1s transgenics develop features diagnostic of human MM, including bone lytic lesions and subendothelial Ig deposition. Furthermore, transcriptional profiles of Eμ-xbp-1s lymphoid and MM cells show aberrant expression of known human MM dysregulated genes. The similarities of this model with the human disease, coupled with documented frequent XBP-1s overexpression in human MM, serve to implicate XBP-1s dysregulation in MM pathogenesis.

Acute myeloid leukemia (AML) is a highly aggressive disease with a high mortality rate that encompasses several genetically and clinically diverse hematological malignancies characterized by clonal expansion of transformed stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of 65 years. Therefore, novel, highly effective therapeutics are needed for this population. Targeting bioenergetic susceptibilities is an exciting area of oncology therapeutics that is potentially applicable in AML. Our group and others have shown that AML blasts depend significantly on mitochondrial oxidative phosphorylation to meet their energy and biomass production demands. Through an extensive medicinal chemistry campaign IACS-10759 was identified as a potent, selective inhibitor of complex I of the electron transport chain with excellent PK and a suitable overall profile. In AML cell lines and primary AML blasts treated ex vivo, we observe a robust decrease in proliferation and a concomitant increase in apoptosis with EC50 values of less than 10 nM. Response to IACS-10759 in AML cells was associated with induction of a metabolic catastrophe that negatively impacted the cells' ability to sustain energy homeostasis, amino acid biosynthesis, and nucleotide production. In a primary AML patient derived xenograft model from a patient who was refractory to standard of care and salvage therapies, 42 days of IACS-10759 (QDx5/week) treatment at 10 mg/kg extended the median survival by greater than 2-fold. Inhibition of OXPHOS by IACS-10759 was confirmed in AML cell lines and PDX models by a decrease in oxygen consumption and significant changes in gene and protein expression, non-essential amino acids and nucleotides. Due to the robust response in AML cell lines, primary AML samples ex vivo, and in vivo efficacy in primary AML PDX models, IACS-10759 has been advanced through IND enabling studies with first-in-human studies targeted for the second quarter of 2016. Citation Format: Jennifer R. Molina, Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Maria Alimova, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Sha Huang, Yongying Jiang, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Timothy McAfoos, Pietro Morlacchi, Melinda Smith, Sonal Sonal, Jay Theroff, Quanyun Xu, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759 is a novel inhibitor of oxidative phosphorylation that selectively targets AML cells by inducing a metabolic catastrophe. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-A15.

Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks needed to enable continued tumor cell growth. Genetic or epigenetic inactivation of one of these two redundant pathways represents a metabolic vulnerability that should be susceptible to an inhibitor of the other pathway. Through an extensive medicinal chemistry campaign, IACS-10759 was identified as a potent inhibitor of complex I of oxidative phosphorylation. In isolated mitochondria or permeabilized cells, ATP production or oxygen consumption was inhibited at single digit nM concentrations in the presence of malate/glutamate, but not succinate. More directly, IACS-10759 inhibited the conversion of NADH to NAD+ in an immunoprecipitated complex I assay at low nM concentrations. Using genetic and pharmacological approaches, the specific complex I subunit inhibited by IACS-10759 has been identified and the mechanism of complex I inhibition is being investigated. Importantly, IACS-10759 is orally bioavailable with excellent physicochemical properties in preclinical species and achieved significant in vivo efficacy with daily oral dosing of 10-25 mg/kg. Specifically, there was a >50 day extension of median survival in an orthotopic AML cell line xenograft and robust regression in DLBCL and GBM xenograft models. In light of these results, as well as its drug like profile IACS-10759 has entered IND enabling studies with first-in-human studies targeted for third quarter of 2015. Citation Format: Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Florian Muller, Timothy Lofton, Timothy McAfoos, Yuting Sun, Melinda Smith, Jay Theroff, Yuanqiang Wu, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-10759: A novel OXPHOS inhibitor which selectively kill tumors with metabolic vulnerabilities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4380. doi:10.1158/1538-7445.AM2015-4380

Acute myeloid leukemia (AML) is a highly aggressive disease that is made up of several genetically and clinically diverse hematological malignancies that are characterized by clonal expansion of malignant stem/progenitor cells with limited ability to differentiate into mature blood cells. Standard of care for AML has progressed minimally in the past 30 years for relapse/refractory AML, with survival rates of 65 years. Therefore, novel, highly effective therapeutics are needed for this population. Growing evidence suggests that in AML, metabolism is altered and that the tumor cells become highly dependent of mitochondria oxidative phosphorylation (OXPHOS) for their survival. We developed IACS-10759 as a novel small molecule inhibitor of complex I that potently inhibits oxygen consumption, eliminates hypoxia, and strongly inhibits the proliferation of cells grown in galactose medium with EC50 values between 1-10 nM. When AML cell lines as well as primary AML cells from relapsed/refractory patients were treated ex vivo with IACS-10759, apoptosis was robustly induced with EC50 values also ranging between 1-10 nM. It is noteworthy, that while apoptosis was induced in primary AML cells, normal patient-derived bone marrow cells were not sensitive. In AML orthotopic xenografts, daily oral dosing with 15 mg/kg IACS-10759 extended median survival to 70 days from 18 days in control animals. Taken together, the robust response in AML cell lines, primary AML samples ex vivo, and efficacy in orthotopic xenografts, IACS-10759 has entered IND enabling studies with first-in-human studies targeted for third quarter of 2015. Citation Format: Marina Protopopova, Madhavi Bandi, Jennifer Bardenhagen, Christophor Bristow, Christopher Carroll, Edward Chang, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Marina Konopleva, Polina Matre, Zhijun Kang, Gang Liu, Florian Muller, Timothy Lofton, Timothy McAfoos, Jay Theroff, Yuting Sun, Yuanqiang Wu, Melinda Smith, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. Relapsed/refractory AML responds robustly to IACS-10759, a novel OXPHOS inhibitor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4455. doi:10.1158/1538-7445.AM2015-4455

OBJECTIVES: The aim of this study was to identify a candidate drug for clinical development from a previously synthesized combinatorial library based on the 1,2-ethylenediamine structure of ethambutol. METHODS: Sixty-nine of the most potent hits against Mycobacterium tuberculosis from the original studies were subjected to a sequential set of tests in vitro and in vivo--determination of MIC for M. tuberculosis H37Rv, cytotoxicity, intracellular antimycobacterial activity, permeability evaluation and in vivo efficacy testing. RESULTS: Twenty-seven compounds with MICs of < or = 15.6 microM were tested on Vero cells to determine in vitro cytotoxicity (IC50) and to establish a selectivity index (SI) (SI = IC50/MIC). Ten compounds with acceptable SI were tested for activity against intracellular bacteria--all were equivalent (within 1%) or superior to ethambutol and several demonstrated cidal activity. Five of the most potent compounds were tested for in vivo efficacy in a murine model of chronic tuberculosis infection. CONCLUSION: Compound SQ109 with an MIC of 0.7-1.56 microM (H37Rv, Erdman and drug-resistant strains of M. tuberculosis), an SI of 16.7 and 99% inhibition activity against intracellular bacteria, demonstrated potency in vivo and limited toxicity in vitro and in vivo, and was selected for further development.

The purpose was to compare the radiation-induced apoptosis in human lymphocytes with DNA-loop relaxation and DNA damage as a function of radiation dose and time after exposure. Morphological changes were analysed by staining with fluorescent dyes and apoptotic fragmentation of DNA with conventional agarose gel electrophoresis, pulsed-field gel electrophoresis (PFGE) and alkaline comet assay. Viability was estimated by trypan blue assay. The levels of protein p53 (TP53) were determined with Western blot. Relaxation of DNA-loops was analysed by the method of anomalous viscosity time dependence (AVTD) and neutral comet assay. Induction and repair of double-strand breaks (DSB) was studied by PFGE and by immunostaining of the TP53 binding protein 1 (53BP1). At various time points of apoptosis, there was a linear dose dependence for all apoptotic end-points up to 1-2 Gy followed by a plateau at higher doses. Immediately after irradiation, relaxation of DNA-loops due to strand breaks was observed. This relaxation had a similar dose-response with saturation at 2-3 Gy. This dose induced approximately one single-strand break (SSB) per 2 Mb of DNA, a value close to the average size of DNA-loops in resting lymphocytes. Similar saturations in dose-responses for apoptosis and DNA-loop relaxation were also observed if cells were treated by camptothecin (CPT) or etoposide VP-16, drugs that relax DNA-loops by induction of SSB and DSB, respectively. The PFGE data showed that the vast majority of DSB were repaired within few hours after irradiation. However, approximately 1.4 foci/Gy/cell, that corresponded to around 3.5% of initial DSB, remained in cells even 24 h after irradiation as measured with immunostaining. The probability to produce one or more than one residual foci per cell was calculated. Radiation at 2-3 Gy induced at least one residual 53BP1 focus per cell. The dose-responses for DNA-loop relaxation, induction of at least one residual 53BP1 foci per cell and apoptosis saturated at 2-3 Gy. The correlation between dose-responses obtained suggested that the DSB in residual foci and relaxation of DNA-loops may be linked to induction of radiation-induced apoptosis in lymphocytes.

Much effort was expended to develop anti-cancer drugs that restore the function of the p53 tumor suppressor protein. However, the p53 activity might be harmful to the organism by amplifying side effects of chemotherapy. Therefore, under certain conditions, inhibition of p53 can serve to prevent inappropriately triggered apoptosis in normal tissues. We have identified a short 22-mer peptide derived from the p53 core domain (peptide 14), which can inhibit p53 specific DNA binding. Upon introduction in living cells, peptide 14 inhibited the ability of p53 to transactivate a reporter gene. Moreover, peptide 14 blocked p53-induced apoptosis in two different cell lines. Peptide 14-mediated inhibition of p53 activity appears to operate via the binding of peptide to the core and/or C-terminal domains of the p53 protein. Our findings provide a basis for the development of a novel approach aimed at the inhibition of p53. This could be essential for the protection from cell death in tissues thus suppressing for example neurodegenerative process or side effects of radio- or chemotherapy.

LY2510924 is a novel selective peptidic CXCR4 antagonist that blocks SDF-1α from binding to its receptor. We have demonstrated that LY2510924 at nanomolar concentrations durably disrupts the SDF-1α/CXCR4 axis in acute myeloid leukemia (AML) cells and exerts anti-leukemia effects as a single agent (AACR 2014: #4768). We further investigated the pronounced anti-leukemia activity of LY2510924 and the mechanisms underlying the anti-leukemia effect. To test the efficacy of LY2510924 in combination with chemotherapy, we injected OCI-AML3/luc/GFP cells into NSG mice. Mice were randomized into 4 groups (10 mice per group) on day 8: control, chemotherapy (cytarabine [50 mg/kg, daily for 5 days, intravenous or intraperitoneal]/doxorubicin [1.5 mg/kg, daily for 3 days, co-delivered intravenously]), LY2510924 (2.5 mg/kg, daily for 3 weeks, subcutaneously), or chemotherapy and LY2510924. Bioluminescence imaging demonstrated that LY2510924 exerted an anti-leukemia effect equal to that achieved with chemotherapy (P=0.249), and the combination therapy group had the lowest luciferase activity. LY2510924-treated mice had prolonged survival (Figure 1) compared to controls (52 days vs. 40 days, p=0.006), and combination therapy extended survival even further (62 days vs. 52 days, p=0.004). Next, we examined anti-leukemia efficacy of LY2510924 in primary human AML xenograft models. NSG mice were injected with primary AML cells and randomized into 2 groups on day 25, after engraftment was documented: control (n=13) and treatment with LY2510924 (n=15; 2.5 mg/kg subcutaneously, daily). First, we examined AML cell mobilization by measuring the proportion of circulating leukemic cells after daily LY2510924 administration. Mice treated with LY2519024 had a significant increase of circulating leukemic cells at 3 hours (2.1-fold, P=0.008), and further increases at 24 hours (2.7-fold, P=0.008) and 48 hours (3.0-fold, P=0.009) compared to controls. Flow cytometry showed a sustained inhibition of CXCR4 12G5 surface expression at 3 and 24 hours after the first LY2510924 injection. Thereafter, weekly examination of circulating leukemic cells in both groups revealed slower progression of leukemia in the LY2510924-treated group (54% vs. 86% circulating AML cells on day 45, P Our previous study (AACR 2014:#4768) demonstrated that LY2510924 did not induce AML cell death in vitro on its own but inhibited AML cell growth in co-cultures with human marrow stromal cells (hMSC). To explore how CXCR4-mediated signaling in AML cells elicits anti-leukemia effects, we performed whole gene expression profiling of FACS-sorted OCI-AML3 cells co-cultured with hMSC for 48 hours and co-treated with LY2510924, in duplicates. Among genes modified by CXCR4 antagonist, we found that CTNNB1 (human beta-catenin), JARID1C (lysine-specific demethylase 5C), RARA (retinoic acid receptor alpha), RARRES2 (chemerin), and COQ4 (coenzyme Q) were downregulated in co-cultured OCI-AML3 cells treated with LY2510925, when compared to either mono-cultured cells or co-cultured cells without LY2510924. These findings are currently being validated by using functional in vitro assays. In conclusion, our findings demonstrate that CXCR4 antagonist LY2510924 inhibits AML progression in leukemia xenograft models in vivo and has a synergistic anti-leukemia effect in combination with chemotherapy. LY2510924 efficiently inhibits CXCR4 signaling in primary AML cells in vivo and induces mobilization of leukemic cells into circulation. This results in pronounced anti-leukemia activity as a single agent. LY2510924's potency and durable occupancy of CXCR4 receptors will likely translate into greater anti-leukemia potency in future clinical applications. Disclosures Peng: Eli Lilly & Company: Employment. Thornton:Eli Lilly & Company: Employment, stocks Other.

Recent studies indicate that acute myeloid leukemia (AML) cells, including leukemia-initiating cells, are highly dependent on oxidative phosphorylation (OXPHOS) for survival, while normal hematopoietic stem cells predominantly utilize glycolysis for energy homeostasis. We have reported development of a series of novel, highly potent mitochondrial complex I inhibitors, which in vitro inhibit complex I with IC50 values Analysis of a panel of AML cell lines showed that a subset of leukemias are markedly dependent on OXPHOS for growth and survival; in this subset, IACS-1131 treatment caused steep decreases in viable cell number via induction of apoptosis. In sensitive cell lines (HL-60, OCI-AML3, KG-1, MV4;11, Kasumi-1), IACS-1131 induced pronounced apoptosis with EC50 between 10 and 100nM, consistent with the IC50 required to inhibit OXPHOS. MOLM13 and OCI-AML2 cells were less sensitive (EC50 250nM and 120nM, and a failure to induce cell death). In primary AML samples from patients with newly diagnosed or relapsed/refractory AML (n=12), the average EC50 for IACS-1131 was 14 ± 8nM in 9 samples, and exceeded 100nM in 3 samples. Consistent with the findings in AML cell lines, 10nM IACS-1131 resulted in partial responses, and 100-250nM resulted in profound loss of viability due to apoptosis induction. In contrast, this treatment caused only a moderate decrease in CD34+ cell numbers and The effects of IACS-1131 on the two major energy-generating pathways, mitochondrial OXPHOS and glycolysis, were investigated using the Seahorse Bioscience XF96 Analyzer. Treatment for 16 hrs caused a striking dose-dependent decrease in basal oxygen consumption rates (OCR), indicating OXPHOS inhibition; reduced ATP production; and decreased maximal respiratory capacity in OCI-AML3 cells and in primary AML blasts (n=9). We confirmed inhibition of complex I in AML cells using Seahorse mitochondrial electron flow assay. These changes preceded changes in viability or apoptotic markers; as such, loss of the mitochondrial membrane potential, annexin V positivity, and induction of mitochondrial reactive oxygen species were seen only at 72 hrs of exposure. Further time-course analysis demonstrated that 2 hrs of IACS-1131 exposure caused significant inhibition of OCR in both sensitive OCI-AML3 and resistant MOLM13 cells, but in MOLM13 cells there was a greater increase in extracellular acidification rates, suggesting compensation by glycolysis. In turn, inhibition of glycolysis with 2-DG, or blockade of pyruvate dehydrogenase kinase with dicholoroacetate (which forces entry into the TCA cycle) sensitized resistant cells to IACS-1131. The intracellular metabolome (polar fraction) of OCI-AML3 cells was characterized following 2, 4, 12 and 24 hrs of treatment with 100nm IACS-1131 using high-resolution magnetic resonance spectroscopy and high mass accuracy Orbitrap mass spectrometry. IACS-1131 modulated levels of the TCA intermediates, producing increased accumulation of citrate and fumarate and decreased succinate and malate, and increased glutathione, possibly because of the oxidative stress. Furthermore, the metabolic analysis indicated a strong effect on amino acid metabolism, whereby IACS-1131 reduced (between 25% and 62% of control) multiple anaplerotic amino acids (including arginine, leucine, isoleucine, valine, phenylalanine, asparagine, histidine, and glutamine, but not aspartate). Finally, IACS-1131 at 60 mg/kg QD po demonstrated robust anti-leukemia activity in an orthotopic OCI-AML3 model. At this dose, IACS-1131 was well tolerated for >50 days and increased median survival duration by more than 5 times (Fig. 1). Studies exploring the anti-AML efficacy of single-agent IACS-1131 in primary AML xenografts are ongoing. Taken together, these data strongly indicate that OXPHOS inhibition constitutes a novel therapeutic approach that targets a unique metabolic vulnerability of AML cells and indicate that further preclinical evaluation of OXPHOS inhibitors is warranted. Figure 1 Figure 1. Figure 1 Treatment with IACS-1131 prolongs survival in a OCI-AML3 xenograft model. Luciferase-expressing OCI-AML3 cells were injected in the tail vein of NSG mice. On day 16 after injection, engraftment was confirmed and mice were randomized on the basis of IVIS-based imaging of luciferase activity after luciferin injection. For the next 58 days, mice received either vehicle or 60 mg/kg/day of IACS-1131 via oral gavage. Mice were sacrificed when body weight was reduced by >20% or for signs of morbidity. Right, bioluminescence imaging before (D0) and 10 days after 1 st dose; left survival. Figure 1. Treatment with IACS-1131 prolongs survival in a OCI-AML3 xenograft model. Luciferase-expressing OCI-AML3 cells were injected in the tail vein of NSG mice. On day 16 after injection, engraftment was confirmed and mice were randomized on the basis of IVIS-based imaging of luciferase activity after luciferin injection. For the next 58 days, mice received either vehicle or 60 mg/kg/day of IACS-1131 via oral gavage. Mice were sacrificed when body weight was reduced by >20% or for signs of morbidity. Right, bioluminescence imaging before (D0) and 10 days after 1 st dose; left survival. Disclosures No relevant conflicts of interest to declare.

Disruption of the SDF-1α/CXCR4 axis by CXCR4 inhibitors has been proven to be an attractive investigational therapeutic approach for acute myeloid leukemia (AML). Moreover, the addition of CXCR4 inhibitor plerixafor to cytotoxic chemotherapy in a phase 1/2 study was associated with increased response rates in relapsed AML (Blood 2012:119;3917). However, plerixafor as a single agent induces only 2- to 4-fold mobilization of leukemic blasts, and this is thought to be due to incomplete inhibition of the SDF-1α/CXCR4 axis and short half-life. LY2510924 is a selective peptidic CXCR4 antagonist that blocks SDF-1α from binding to the receptor. Flow cytometry using OCI-AML3 cells showed that LY2510924 inhibited binding of anti-CXCR4 antibody 12G5 to surface CXCR4 in a concentration-dependent fashion. LY2510924 was 100 times more potent compared to plerixafor (normalized surface expression, LY2510924 at 1 nM, 10.7±0.27%; plerixafor at 1 nM and 100 nM, 74.6±1.23% and 11.0±0.29%, respectively). The action of LY2510924 started as early as 1 minute and continued up to 72 hours at 10 nM. SDF-1α induced migration of OCI-AML3 and primary AML cells, which was abolished by 1 nM LY2510924 but not suppressed by 1 nM plerixafor. LY2510924 inhibited SDF-1α-induced AKT and/or ERK phosphorylation in AML cell lines (OCI-AML3 and U937) and primary AML cells as shown by immunoblotting and multi-parameter phospho-flow cytometry. To test the efficacy of LY2510924 in vivo, we injected OCI-AML3/luc/mCherry cells into sub-lethally irradiated NSG mice. Twelve days after cell injection, mice were randomized into 4 groups: control, cytarabine, LY2510924, and LY2510924 plus cytarabine. On day 21 after cell injection, we observed a 3.4±1.4-fold increase of circulating leukemic cells at 3 hours and a 24.1±15.4-fold mobilization at 24 hours after LY2510924 injection. Bioluminescence imaging demonstrated that mice treated with LY2510924 had lower leukemia burden than cytarabine-treated and control mice. On day 39, the combination group showed less luciferase activity than the LY2510924 group (P=0.034). Hematoxylin-eosin and immunohistochemical staining with human CD45 antibody demonstrated that the LY2510924-treated groups had a significant decrease in leukemic infiltration of bone marrow, spleen, liver, and lung. This anti-leukemia effect translated into a significant prolongation of survival in LY2510924-treated mice (40 days vs. 26 days, P In summary, in vitro data showed that a novel peptidic CXCR4 antagonist, LY2510924, could effectively disrupt the SDF-1α/CXCR4 axis in AML cells and was more potent than plerixafor. In vivo data showed anti-leukemia effects of LY2510924 as a single agent. LY2510924's stronger potency than plerixafor, rapidity of action, and prolonged CXCR4-inhibitory effects will likely translate into a higher anti-leukemia potency than that of plerixafor in future clinical applications. Citation Format: Byung Sik Cho, Zhihong Zeng, Hong Mu, Teresa McQueen, Marina Protopopova, Jorge Cortes, Joe Marszalek, Sheng-Bin Peng, Donald E. Thornton, Michael Andreeff, Marina Konopleva. Novel peptidic CXCR4 antagonist LY2510924 disrupts the SDF-1α/CXCR4 axis resulting in anti-AML efficacy in vivo. [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 4768. doi:10.1158/1538-7445.AM2014-4768

Inhibition of mitochondria complex I in tumors that are metabolically dependent on oxidative phosphorylation (OXPHOS) for their survival offers unique synthetic lethal opportunities. Examples of dependent contexts are AML and DLBCL, where OXPHOS is highly active and subpopulations of glioblastoma and neuroblastoma that possess genetic alterations which make them glycolysis deficient. In addition, several lines of evidence indicate that after treatment with chemo or targeted therapy, residual tumor cells become reliant on OXPHOS for their continued survival. In each of these cellular states, excessive dependence on OXPHOS renders tumor cells vulnerable to therapeutic targeting strategies that exploit this addiction. We have generated a series of novel, highly potent complex I inhibitors, which in vitro inhibit complex I with IC50 values < 10 nM. When tested in cultured cell systems (cell lines and spheroids) these compounds inhibit oxygen consumption, eliminate hypoxia, and strongly inhibit the proliferation cells grown in galactose medium with EC50 values between 1-10 nM. Lead compounds specifically induce apoptosis with EC50 values between 1-10 nM in OXPHOS dependent cancer models such as AML and DLBCL cell lines and in glycolysis deficient cancer cell lines. Of note, apoptosis is induced in primary AML cells but not in normal patient-derived CD34+ cells. These compounds are orally bioavailable with excellent pharmacokinetics properties in preclinical species making them appropriate tools for proof-of-concept studies in vivo. In agreement with data in cell culture, we have shown that daily oral treatment with as low as 5 -10 mg/kg of our OXPHOS inhibitors is well tolerated and induce strong regression of NB-1 (glycolysis-deficient cells) subcutaneous and intracranial xenografts. We have also demonstrated that sustained pharmacological inhibition of OXPHOS induce regression of DLBCL subcutaneous models and dramatically increase mice survival in an OCI-AML3 orthotopic xenograft model. In addition to synthetic lethality in monotherapy, we are exploring whether OXPHOS inhibition can overcome resistance to radiotherapy, chemotherapy and specific targeted therapies. Taken together, these data strongly support the notion that inhibiting OXPHOS in hypersensitive populations could be a novel, innovative therapeutic approach and justifies evaluation of OXPHOS inhibitors in a clinical setting. Citation Format: Joseph R. Marszalek, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Ninping Feng, Barbara Czako, Jason Gay, Mary Geck Do, Jennifer Greer, Ryan M. Johnson, Marina Konopleva, Zhijun Kang, Gang Liu, Timothy Lofton, Timothy McAfoos, Marina Protopopova, Alessia Petrocchi, Florian Muller, Jay Theroff, Yuanqing Wu, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, Emilia Di Francesco. Identification of OXPHOS inhibitors which selectively kill tumors with specific metabolic vulnerabilities. [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 949. doi:10.1158/1538-7445.AM2014-949

Recent evidence identified a genetic and functional link between Chk2 kinase and p53 as a candidate genome integrity checkpoint and a tumour suppressor pathway. Here we report that in human cells, Chk2 and p53 form protein-protein complexes whose abundance increased upon DNA damage, and whose formation was abrogated through cancer associated mutations in the FHA domain of Chk2, or mutations in the tetramerization domain of p53. Whereas among Li-Fraumeni syndrome families mutations of Chk2 or p53 occur in a mutually exclusive manner, we document that the colon cancer cell line HCT-15 concomitantly lacks functions of both Chk2 and p53, the latter demonstrated by a non-invasive reporter assay monitoring p53-dependent transactivation in live cells. Despite the preserved ability of common cancer-derived mutant p53 proteins to bind and potentially 'titrate' activated Chk2, the integrity of the S phase checkpoint response to ionizing radiation remained largely intact and dependent on Chk2 in cells with wild-type, mutant, or no p53. These results provide new mechanistic insights into the Chk2-p53 interplay, suggest how mutations in Chk2 may abrogate its tumour suppressor function, and indicate that compared with individual defects in either Chk2 or p53, concomitant mutations in both of these cell cycle checkpoint regulators may provide some additional selective advantage to tumour cells.

In addition to binding DNA in a sequence-specific manner, the p53 tumour suppressor protein can interact with damaged DNA. In order to understand which structural features in DNA the C-teminal domain recognises we have studied the interaction of p53 protein with different types of DNA oligonucleotides imitating damaged DNA. Here we show that one unpaired nucleotide within double-stranded (ds)DNA is sufficient for recognition by the p53 C-terminus, either as a protruding end or as an internal gap in dsDNA. C-terminal interaction with DNA ends facilitated core domain binding to DNA, whereas interaction with gaps prevented core domain-DNA complexing, implying that p53 might adopt distinct conformations upon binding to different DNA lesions. These observations suggest that both single-strand and double-strand breaks can serve as a target for p53 C-terminal recognition in vivo and indicate that p53 might recruit different repair factors to the sites of damaged DNA depending on the type of lesion.

The transcription factor X-box binding protein-1 (XBP-1) plays critical roles in the unfolded protein response (UPR), the differentiation of plasma cells, and the regulation of growth factor signaling pathways. XBP-1 is subject to regulation by alternative RNA processing producing XBP-1-spliced (S) and -unspliced (U) mRNAs encoding proteins with identical DNA-binding and bZIP domains yet distinct C-terminal transactivation domains. The weaker transactivation potential of XBP-1(U) has prompted speculation that it may influence XBP-1(S) activity as a transdominant mutant. While elevated XBP-1 expression has been reported in transformed cells, the relative ratios of these XBP-1 isoforms and associated physiological relevance in cancer are uncertain. Here, we assessed the differential impact of enforced XBP-1(S) versus XBP-1(U) transgene expression in the B cell lineage. Both transgenes elicited early onset antibody-dependent autoimmune disease characterized by elevated levels of serum immunoglobulin (Ig) and IL-6 production, increased numbers of marginal zone and mature follicular B cells in the spleen, and expanded mature B cell populations in the bone marrow. Notably, aged XBP-1(S) mice developed clonal plasma cell expansions, culminating in the human-equivalent of Monoclonal Gammopathy of Undetermined Significance (MGUS) or Multiple Myeloma (MM). Conversely, XBP-1(U) mice develop multi-organ lymphoplasmacytic infiltrates and, with advancing age, succumb to neoplasms resembling human Lymphoplasmacytic Lymphoma/Waldenstrom’s Macroglobulinemia (LPL/WM). These unanticipated genetic observations in the mouse were translated to human disease with documentation of elevated levels of XBP-1(S) in MM and XBP-1(U) in LPL/WM. Together, these results indicate that imbalances in XBP-1(S) and XBP-1(U) alters B cell lineage homeostasis and can drive two distinct types of lymphoplasmacytic neoplasms in vivo. The findings of this study, together with the known capacity of XBP-1 to regulate various cancer-relevant growth factor signaling pathways, predicts that epigenetic dysregulation of alternate XBP-1 RNA processing can promote age-associated B cell malignancies in humans.

Short abstracts of theses on oncologic subjects are published under this heading. The abstract should contain background, problems, results and conclusions and be an independent informative unit that can be read without access to the thesis. It should not contain references to literature, figures or tables in the thesis. A suitable size is about 500 words. The abstract can be sent to Acta Oncologica together with information about department, faculty and university and date of dissertation.

Multiple Myeloma (MM) is characterized by a clonal proliferation of abnormal plasma cells in the bone marrow and is among the most frequent and lethal hematological diseases. In spite of significant effort towards the identification of the molecular events leading to this malignancy, the genetic alterations responsible for the pathogenesis of this disease remain poorly understood. Regional copy number alterations (CNAs) in cancer genomes have been among the most informative structural changes in cancer and have led to the discovery of many oncogenes and tumor supressor genes. Using array comparative genomic hybridization (array-CGH) and expression microarray technologies we have analyzed a large collection of cell lines and clinically annotated primary tumors. This high-resolution genomic analysis has identified all previously reported regional gains and losses as well as many novel highly recurrent genetic loci with potential biological and clinical relevance. In particular, we have identified an amplification at chromosome 1q21 as one of the most recurrent genetic changes in cell lines and in a subgroup of primary tumors. This chromosomal change has been previously implicated with disease progression. Analysis across several cell lines has allowed the identification of a Minimal Common Region (MCRs) of amplification at 1q21. Correlation between DNA copy number changes and expression profiling data has identified a limited set of candidate genes within this MCR that are amplified and overexpressed. Using shRNAi technology we have identified BCL-9 as a candidate gene residing at the 1q21 MCR. In vitro and in vivo functional data about the role of BL-9 will be presented. These data will provide critical understanding on the diverse pathways leading to Multiple Myeloma progression.