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Puromycin is an amino nucleoside that inhibits protein synthesis by interrupting elongation of nascent peptide chains. It is a commonly used selection antibiotic in molecular biology research via engineered expression of a puromycin resistance transgene. The enzyme puromycin acetyl transferase (pac) or PuroR inactivates puromycin by N-acetylating its reactive amino group. Puromycin acetylation by pac requires the central metabolite and acetyl group donor acetyl-CoA as a substrate. We found that puromycin treatment exacerbates sensitivity of cancer cells to knockdown of pantothenate kinases, the proteins that catalyze the rate-limiting step of de novo coenzyme A production in cells. Mechanistically, we found that ablation of PANKs together with puromycin depletes acetyl-CoA levels, in a manner modulated by the dose of puromycin. Our findings provide a note of caution and context in the use of puromycin for metabolism research in that interference with the major acyl donor used for inactivating biotransformation may exacerbate toxicity under selection. Broadly, our findings also invite studies to explore how targeting CoA and acetyl-CoA synthesis may be exploited to enhance cytotoxic effects of cancer drugs that undergo acetylation.

Oncogenic mutations in the KRAS oncogene hyperactivate the MAPK pathway, a key driver of oncogenesis in the majority of human malignancies. KRAS-driven tumors are aggressive and highly refractory to standard-of-care treatments, thus intense efforts have been devoted to developing inhibitors of its activity. However, pioneering studies in engineered conditional mKras driven mouse models of pancreatic ductal adenocarcinoma (KPC) have demonstrated that while mKras is necessary for tumor maintenance, the anti-neoplastic effects of mKras extinction can be counteracted by activation of the Hippo pathway. These data strongly suggest that the full realization of MAPK pathway (including KRAS) inhibitors’ clinical potential requires the concomitant suppression of the function of YAP/TEAD, the effectors of Hippo pathway. Here, we directly test this hypothesis utilizing Sporos BioDiscovery’s next generation TEAD inhibitor (SPR1) - optimized to maximize anti-neoplastic activity and minimize toxicity through fine-tuning of TEAD paralog specificity. Using mKras murine (KrasG12D mutant, p53 null, PDAC) and human cell lines - we demonstrated strong interaction between SPR1 TEAD inhibitors and the clinical MEK inhibitors (since no G12D inhibitor is yet approved) Trametinib/Binimetinib in vitro. Functional TEAD inhibition was also shown by the reduced expression of the YAP/TEAD transcriptional targets CTGF and CYR61. Further, using a well-established murine orthotopic model, KPC (KrasG12D;p53-/-;p48Cre), that faithfully mimics human PDAC, we demonstrated robust combined efficacy of Trametinib and SPR1 TEAD inhibitors in vivo in suppressing tumor growth and strikingly doubling the survival time of the tumor-bearing mice. To our knowledge, this is the first preclinical demonstration of the therapeutic potential of a MAPK + TEAD inhibitor combination in a mKras G12D-driven orthotopic PDAC; given the high barrier for efficacy of this KPC model –we believe these results favorably presage generalization to MAPK/TEAD inhibitor combinations in other cancer setting and translatability to the clinic. Citation Format: Deepavali Chakravarti, Florian Muller, Jeno Gyuris, Erkun Baloglu, Selvi Kunnimalayiaan, Jill Olson, Sharon Shacham. Combined inhibition of the MAPK and Hippo pathways drives efficacious tumor suppression in a faithful model of mutant Kras (KPC) PDAC [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B003.

Sulfation of proteins, carbohydrates, lipids, and xenobiotics is an essential post-translational modification (PTM) process thought to play critical roles in diverse biological processes ranging from detoxification, cell signaling, and extracellular matrix architecture, to immune modulation. Sulfation is accomplished by the universal sulfate donor, PAPS (3'-Phosphoadenosine-5'-phosphosulfate), which is synthesized by bifunctional enzymes PAPSS1 and PAPSS2 (PAPS synthases). The PAPSS2 gene situates near PTEN and is frequently deleted with PTEN across cancer types. Approximately 20% of prostate cancer patients exhibit loss of PTEN, and ~50% of these cases also sustain a loss of PAPSS2. However, the loss of PAPSS2 appears to be tolerated and possibly compensated by its functionally redundant paralogue, PAPSS1, located on chromosome 4q24. The functional redundancy between PAPSS1 and PAPSS2 suggests that these two genes may be collateral lethality pair provided that sulfation is essential for cancer cell viability. Thus, we hypothesize that targeting PAPSS1 in PTEN/PAPSS2-null prostate cancer can generate cancer-specific vulnerabilities while leaving normal cells undisturbed. To assess this possibility, knockdown and knockout of PAPSS1 in cell lines of PAPSS2-null and PAPSS2-wildtype background were generated to characterize cell viability in vitro and tumor formation in vivo. PAPS and APS, an intermediary product of the sulfation pathway, are measured to verify that no alternative pathways for sulfate donors exist and that the co-extinction of PAPSS1/2 eliminates all avenues of generating sulfate donors. Combined extinction of PAPSS1/2 across multiple cancer cell lines was shown to be tolerated in vitro, and recurrent changes in morphology were observed. Loss of sulfation verified by the disappearance of sulfotyrosine and mass spectrometry measurements of PAPS and APS are pending. Our In vitro results surprisingly indicate that a major PTM, like sulfation is entirely dispensable for cancer cell viability under normal culture conditions. However, PAPSS1/2-null cell lines demonstrated a profound delay in tumor formation and prolonged survival, suggesting that sulfation may be required for stromal and innate immune modulation. Citation Format: Ko-Chien Chen, Yonhong Liu, Chenchu Lin, Er-Yen (Nick) Yen, Francesca Citron, Xingdi Ma, Tan Lin, Philip Lorenzi, Florian Muller, Ronald DePinho. Sulfation is required for prostate cancer xenograft tumor formation but is dispensable for cell viabilityin vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6053.

Monolithic crystals are examined as an alternative to segmented scintillator arrays in positron emission tomography (PET). Monoliths provide good energy, timing and spatial resolution including intrinsic depth of interaction (DOI) encoding. DOI allows reducing parallax errors (radial astigmatism) at off-center positions within a PET ring. We present a novel DOI-estimation approach based on the supervised machine learning algorithm gradient tree boosting (GTB). GTB builds predictive regression models based on sequential binary comparisons (decision trees). GTB models have been shown to be implementable in FPGA if the memory requirement fits the available resources. We propose two optimization scenarios for the best possible positioning performance: One restricting the available memory to enable a future FPGA implementation and one without any restrictions. The positioning performance of the GTB models is compared with a DOI estimation method based on a single DOI observable (SO) comparable to other methods presented in literature. For a 12 mm high monolith, we achieve an averaged spatial resolution of 2.15 mm and 2.12 mm FWHM for SO and GTB models, respectively. In contrast to SO models, GTB models show a nearly uniform positioning performance over the whole crystal depth., The accepted version of the article; DOI included; added one citation; results kept unchanged

Angiogenesis inhibition has become a mainstay of oncology despite having fallen short of its early promise. As originally envisioned, angiogenesis inhibition would cut off the blood supply, deprive tumor cells of key nutrients, leading to their demise. In practice, while there is evidence that tumors under angiogenesis treatment do in fact exhibit some degree of metabolic stress, this is stress is not sufficient to induce significant cancer cell death. We posit that the full potential of angiogenesis inhibition can be realized by the combination of angiogenesis inhibition with emerging tumor metabolism targeting therapies. Because tumors under angiogenesis inhibition are already in a state of nutrient stress, the effects of metabolically targeted therapies such as amino acid depletion (e.g. asparginase, methionine restriction), inhibitors of stress adaption (AMPK and GCN2 inhibitors) or energy metabolism (e.g. IACS-010759, Metformin, POMHEX) stand to dramatically increase in potency whilst remaining selective for (angiogenic) tumor versus (non-angiogenic) normal tissue. Here, we provide proof-of-principal for this thesis. First, we performed metabolomic profiling of angiogenesis-inhibited tumors, which corroborates a state of nutrient stress in angiogenesis-inhibited tumors. Second, we demonstrate dramatic anti-neoplastic synergy (effectively curing of xenografted tumor-bearing mice, irrespective of initial tumor size), without enhanced adverse toxicities, between the anti-VEGFA antibody Avastin and enolase inhibitor HEX. The same results were recapitulated with the VEGFR inhibitor Tivozanib and HEX and the enolase inhibitor could be substituted with the Oxphos inhibitor IACS-010759, with similar effects. The synergy was observed in a broad range of tumor types, even those without clear genetic susceptibilities. Together, these results suggest that angiogenesis inhibitors synergize broadly with cancer therapies targeting metabolism, allowing the realization of the full potential of these previously disappointing drugs. Our results warrant systematic combination clinical trials between angiogenesis inhibitors and established, as well as emerging anti-metabolic cancer therapies. Citation Format: Sunada Khadka, Yu-Hsi Lin, Jeffery Ackroyd, Kenisha Arthur, Yasaman Barekatain, Anton Poral, Theresa Tran, Eliot Behr, Yining Chen, Dimitra Georgiou, Ronald Depinho, Florian Muller. Angiogenesis inhibitors strongly synergize with therapeutics targeting tumor metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3208.

Homozygous deletion of the CDK2NA locus frequently results in the co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiform (GBM). In cell culture, cell lines with MTAP-deletions show elevations of its substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit PRMT5, which sensitizes MTAP-deleted cell lines to PRMT5 and MAT2A inhibition. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAPhomozygous deleted GBM. Citation Format: Yasaman Barekatain, Jeffrey Ackroyd, Victoria Yan, Sunada Khadka, Ko-Chien Chen, Raghu Kalluri, John de Groot, Jason Huse, Florian muller. Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2395.

Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates by oxidation of anaplerotic substrates, such as glucose-derived pyruvate and glutamine20 derived glutamate. We have previously documented that a subset of tumors with 1p36 homozygous deletion exhibit co-deletion of ENO1, in turn becoming extremely dependent on its redundant isoform ENO2 and sensitive to an overall enzymatic deficiency of enolase. Metabolomic profiling of ENO1-deleted glioma cells treated with an enolase inhibitor revealed a profound decrease in TCA cycle metabolites, which correlated with cell-line specific sensitivity to enolase inhibition, highlighting the importance of glycolysis derived pyruvate for anaplerosis. Correspondingly, the toxicity of the enolase inhibitor was significantly attenuated by exogenous supplementation of supraphysiological levels of anaplerotic substrates including pyruvate. These findings led us to hypothesize that cancer cells with ENO1 homozygous deletions treated with an enolase inhibitor might show exceptional sensitivity to inhibition of glutaminolysis because of reduced anaplerotic flow from glycolysis. We found that ENO1-deleted cells indeed exhibited selective sensitivity to the glutaminase inhibitor CB-839, and this sensitivity was also attenuated by exogenous supplementation of anaplerotic substrates including pyruvate. Despite these promising in vitro results, the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Together, these data suggest that at least for 1p36-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of the in vitro and in vivo environments as the potential reasons for the differential efficacy of metabolism targeted therapies in in vitro and in vivo systems.

Ongoing compilation of data supporting clinical advancement of GS-441524, the parent nucleoside of remdesivir, for the treatment of Covid-19. Version 3. Last updated: Aug. 29, 2020.

While remdesivir has garnered much hope for its moderate anti-Covid-19 effects in recent clinical trials, its parent nucleoside, GS-441524 has remained out of the spotlight despite exhibiting comparable potency in clinically relevant models of the lung. Our analysis of the in vivo pharmacokinetics of remdesivir evidences premature of hydrolysis of its phosphate pro-drug in serum such that GS-441524 is the predominant circulating metabolite that reaches the lungs. Under this broader pharmacokinetic rationale, we contend that GS-441524 is superior to remdesivir for Covid-19 treatment due to its synthetic simplicity, demonstrated in vivo potency against coronavirus models, and comparative ease of formulation into an inhalable prophylactic. Collectively, these advantages would simplify mass production and distribution and enable higher dosing of GS-441524 both therapeutically and prophylactically.

Glycolysis inhibition remains aspirational in cancer therapy. We recently described a promising phosphonate inhibitor of Enolase for cancers harboring deletions in ENO1. Here, we describe the application of nitroheterocycle pro-drugs capitalizing on tumor hypoxia. These bioreducible pro-drugs exhibit up to 14-fold greater potency under hypoxic conditions compared to normoxia and exhibit robust stability in biological fluids. Our work provides strong proof-of-concept for using bioreduction as a pro-drug delivery strategy in the context of Enolase inhibition.

International audience; The analysis of fuel rod bundle flows constitutes a key element of Pressurized-Water Reactors (PWR) safety studies. The present work aims at improving our understanding of nefarious reorganisation phenomena observed by numerous studies in the flow large-scale structures. 3D simulations allowed identifying two distinct reorganisations consisting in a sign change for either a transverse velocity in rod-to-rod gaps or for a subchannel vortex. A Taylor “frozen turbulence” hypothesis was adopted to model the evolution of large-scale 3D structures as transported-2D. A statistical method was applied to the 2D field to determine its thermodynamically stable states through an optimization problem. Similarities were obtained between the PWR coherent structures and the stable states in a simplified 2D geometry. Further, 2D simulations allowed identifying two possible flow bifurcations, each related to one of the reorganisations observed in 3D simulations, laying the foundations for a physical explanation of this phenomenon.

IEEE transactions on radiation and plasma medical sciences : ITRPFI 2(5), 411-421 (2018). doi:10.1109/TRPMS.2018.2837738, Published by IEEE, New York, NY

Cardiac glycosides (CGs) are approved for the treatment of cardiovascular alterations and their known cellular target is the alpha subunit of the sodium (Na+)/potassium (K+)-ATPase (NKA). Pharmacologically, they represent a well-known generation of drugs for treating cardiovascular problems, thus allowing the investigation of potential dose-dependent side effects. Interestingly, since the end of the 1960s, epidemiological studies have indicated that anti-cancer effects were associated with the regular use of these compounds. Since then, a large body of evidence has been accumulated on the in vitro and in vivo effects of CGs in various experimental models, thus confirming their selective action on cancer cell proliferation and viability. CGs have the potential for targeted therapeutic applications. Many of the anti-cancer activities of these compounds have been linked to the inhibition of their primary target, the NKA. A number of studies have shown a correlation between the overexpression of specific alpha subunits in cancerous versus non-cancerous cells and cancer cell responsiveness. Other findings have provided evidence of the on-target nature of the ascribed anti-cancer effects. More recently, studies have indicated additional intracellular targets for these agents, whose modulation might be, at least in some instances, unrelated to NKA targeting. These include endosomal trafficking of both NKA and Src kinase, downregulation of pro-survival Mcl-1 and Bcl-xL pro-survival proteins, and immunogenic cell death induction, among others. This research update summarizes the current knowledge about CGs as new, targeted anti-cancer agents, alone or in combination with other chemotherapeutic compounds.

Accelerated glycolysis is the main metabolic change observed in cancer, but the underlying molecular mechanisms and their role in cancer progression remain poorly understood. Here we show that deletion of the long noncoding RNA (lncRNA) Neat1 in MMTV-PyVT mice profoundly impairs tumor initiation, growth and metastasis, while specifically switching off the penultimate step of glycolysis. Mechanistically, NEAT1 directly binds and forms a scaffold bridge for the assembly of PGK1/PGAM1/ENO1 complexes, and thereby promotes substrate channeling for high and efficient glycolysis. Notably, NEAT1 is upregulated in cancer patients and correlates with high levels of these complexes, and genetic and pharmacological blockade of penultimate glycolysis ablates NEAT1-dependent tumorigenesis. Finally, we demonstrate that Pinin mediates glucose-stimulated nuclear export of NEAT1, through which it exerts isoform-specific and paraspeckle-independent functions. These findings establish a direct role for NEAT1 in regulating tumor metabolism, provide new insights into the Warburg effect, and identify potential targets for therapy.

The localization of messenger RNA (mRNA) molecules inside cells play an important role for the local control of gene expression. However, the localization patterns of many mRNAs remain unknown and poorly understood. Single Molecule Fluorescence in Situ Hybridization (smFISH) allows for the visualization of individual mRNA molecules in cells. This method is now scalable and can be applied in High Content Screening (HCS) mode. Here, we propose a computational workflow based on deep convolutional neural networks trained on simulated data to identify different localization patterns from large-scale smFISH data.

Lung cancer is a devastating disease that remains a top cause of cancer mortality. Despite improvements with targeted and immunotherapies, the majority of patients with lung cancer lack effective therapies, underscoring the need for additional treatment approaches. Genomic studies have identified frequent alterations in components of the SWI/SNF chromatin remodeling complex including SMARCA4 and ARID1A. To understand the mechanisms of tumorigenesis driven by mutations in this complex, we developed a genetically engineered mouse model of lung adenocarcinoma by ablating Smarca4 in the lung epithelium. We demonstrate that Smarca4 acts as a bona fide tumor suppressor and cooperates with p53 loss and Kras activation. Gene expression analyses revealed the signature of enhanced oxidative phosphorylation (OXPHOS) in SMARCA4 mutant tumors. We further show that SMARCA4 mutant cells have enhanced oxygen consumption and increased respiratory capacity. Importantly, SMARCA4 mutant lung cancer cell lines and xenograft tumors have marked sensitivity to inhibition of OXPHOS by a novel small molecule, IACS-010759, that is under clinical development. Mechanistically, we show that SMARCA4-deficient cells have a blunted transcriptional response to energy stress creating a therapeutically exploitable synthetic lethal interaction. These findings provide the mechanistic basis for further development of OXPHOS inhibitors as therapeutics against SWI/SNF mutant tumors.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

Molecules targeting pro-inflammatory pathways have demonstrated beneficial effects in cancer treatment. More recently, combination of natural and synthetic anti-inflammatory drugs was suggested as an appealing strategy to inhibit tumor growth. Herein, we show that curcumin, a polyphenol from Curcuma longa and celecoxib induce apoptosis in hematopoietic cancer cell lines (Hel, Jurkat, K562, Raji, and U937). Further investigations on the most sensitive cell line, U937, indicated that these effects were tightly associated with an accumulation of the cells in S and G2/M for curcumin and in G0/G1 phase of cell cycle for celecoxib, respectively. The effect of celecoxib on cell cycle is associated with an induction of p27 and the down-regulation of cyclin D1. However, in the case of combination experiments, the pretreatment of U937 cells with celecoxib at non-apoptogenic concentrations counteracted curcumin-induced apoptosis. We found that this effect correlated with the prevention of the accumulation in S and G2/M phase of cell cycle induced by curcumin. Similar results have been obtained when celecoxib and curcumin were co-administrated at the same time. Overall our data suggest that this natural and synthetic drug combination is detrimental for cell death induction.

In the version of this article originally published, information regarding several funding sources was omitted from the Acknowledgements section. The following sentences should have been included: "This work was supported by the generous philanthropic contributions to The University of Texas MD Anderson Lung Cancer Moon Shots Program, the UT Lung SPORE 5 P50 CA07090, and the MD Anderson Cancer Center Support Grant P30CA01667. V.P is supported by R01CA155196-01A1 from the National Cancer Institute." Also, reference 18 was incorrect. The original reference was: Kim, E. S. et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov. 1, 44-53 (2011). It should have been: Papadimitrakopoulou, V. et al. The BATTLE-2 study: a biomarker-integrated targeted therapy study in previously treated patients with advanced non-small-cell lung cancer. J Clin. Oncol. 34, 3638-3647 (2016). The errors have been corrected in the HTML and PDF versions of this article.

Over the past few years we and others reported that specific populations of tumor cells including AML, subsets of lymphoma, glioblastoma, triple negative breast cancer (TNBC), melanoma and pancreatic ductal adenocarcinoma (PDAC) are highly dependent upon oxidative phosphorylation (OXPHOS) to meet their energy and biomass needs. Inhibition of OXPHOS in the context of these dependent tumor populations represents therefore an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign we discovered IACS-010759, a potent, selective small molecule inhibitor of complex I of the mitochondria electron transport chain that possesses excellent pharmacokinetic (PK) and pharmacologic properties, making it suitable for clinical development. We advanced IACS-010759 through IND studies and have recently initiated Phase I studies in patients with relapsed/refractory acute myeloid leukemia (AML) and advanced solid tumors and lymphomas (NCT02882321 and NCT03291938). In this presentation we will describe the identification of a novel series of Complex I inhibitors and their optimization into the clinical candidate compound IACS-010759. Several challenges were successfully overcome, including the optimization of the pharmacokinetic profile and the identification of inhibitors with minimal activity shift across preclinical species, thus enabling a thorough evaluation of the efficacy and toxicology profile. Aspects of the extensive translational research conducted to elucidate the mechanism of action of IACS-010759 and to position it into the clinic will be discussed, including the compelling pharmacological response observed in multiple PDX models of primary AML, and PDX xenograft models of lymphoma, TNBC, glioblastoma, melanoma and PDAC. The observed response was associated with robust pharmacodynamic read-out as assessed by modulation of oxygen consumption rate (OCR), aspartate and specific transcriptional changes. The presentation will also cover the preclinical development activities which resulted in IACS-010759 advancing into on-going phase 1 evaluation in AML and solid tumors. Citation Format: Maria Emilia Di Francesco, Joseph R. Marszalek, Timothy McAfoos, Christopher L. Carroll, Zhijun Kang, Gang Liu, Jay P. Theroff, Jennifer P. Bardenhager, Madhavi L. Bandi, Jennifer R. Molina, Sonal Gera, Marina Protopopova, Yuting Sun, Mary K. Geck Do, Ningping Feng, Jason P. Gay, Florian Muller, Marina Konopleva, Funda Meric-Bernstam, Carlo Toniatti, Timothy P. Heffernan, Giulio F. Draetta, Philip Jones. Discovery and development of IACS-010759, a novel inhibitor of Complex I currently in phase I studies to exploit oxidative phosphorylation dependency in acute myeloid leukemia and solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1655.

In this paper we introduce a new approach for improving hand-guiding of robotic arms. We present the tunnel-shaped potential force field (TSPFF) which guides the user along a reference trajectory in order to improve the user comfort of handling robots. The reference trajectory is computed using the learning data of experienced users. The TSPFF is realized by an impedance control of the robot. The main focus of this paper is to find a robust parametrization of the TSPFF. For this purpose the stability of user-robot interaction is analyzed by means of the Lyapunov stability theory. The results are complemented by simulation studies in which the influence of the parametrization on control performance and user comfort is analyzed. The resulting rules for parametrization are validated by experimental studies with a robot with six degrees of freedom.

Cancer development is mostly due to a deregulation of cell death as cancer cells become resistant to apoptosis by increasing expression of anti-apoptotic proteins belonging to the Bcl-2 family. Mcl-1 is one anti-apoptotic protein, which is mainly responsible for cancer cell resistance as it is overexpressed by most cancer cell types. Many research projects aim to restore cancer cell death by using natural pharmacological scaffolds targeting anti-apoptotic proteins to inhibit their effect in cancer development. This review introduces natural compound inhibitors of the Bcl-2 protein family with a focus on Mcl-1.

Cyclooxygenase-2 (COX-2) is an essential regulator of cancer promotion and progression. Extensive efforts to target this enzyme have been developed to reduce growth of cancer cells for chemopreventive and therapeutic reasons. In this context, cyclooxygenase-2 inhibitors present interesting antitumor effects. However, inhibition of COX-2 by anti-COX-2 compounds such as celecoxib was recently associated with detrimental cardiovascular side effects limiting their clinical use. As many anticancer effects of celecoxib are COX-2 independent, analogs such as 2,5-dimethyl-celecoxib (DMC), which lacks COX-2-inhibitory activity, represent a promising alternative strategy. In this study, we investigated the effect of this molecule on growth of hematologic cancer cell lines (U937, Jurkat, Hel, Raji, and K562). We found that this molecule is able to reduce the growth and induces apoptosis more efficiently than celecoxib in all the leukemic cell lines tested. Cell death was associated with downregulation of Mcl-1 protein expression. We also found that DMC induces endoplasmic reticulum stress, which is associated with a decreased of GRP78 protein expression and an alteration of cell cycle progression at the G1/S transition in U937 cells. Accordingly, typical downregulation of c-Myc and cyclin D1 and an upregulation of p27 were observed. Interestingly, for shorter time points, an alteration of mitotic progression, associated with the downregulation of survivin protein expression was observed. Altogether, our data provide new evidence about the mode of action of this compound on hematologic malignancies.

Cells contain many subcellular structures in which specialized proteins locally cluster. Binding interactions within such clusters may be analyzed in live cells using models for fluorescence recovery after photobleaching (FRAP). Here we analyze a three-dimensional FRAP model that accounts for a single spatially localized cluster of binding sites in the presence of both diffusion and impermeable boundaries. We demonstrate that models completely ignoring the spatial localization of binding yield poor estimates for the binding parameters within the binding site cluster. In contrast, we find that ignoring only the restricted axial height of the binding-site cluster is far less detrimental, thereby enabling the use of computationally less expensive models. We also identify simplified solutions to the FRAP model for limiting behaviors where either diffusion or binding dominate. We show how ignoring a role for diffusion can sometimes produce serious errors in binding parameter estimation. We illustrate application of the method by analyzing binding of a transcription factor, the glucocorticoid receptor, to a tandem array of mouse mammary tumor virus promoter sites in live cells, obtaining an estimate for an in vivo binding constant (10(-7) M), and a first approximation of an upper bound on the transcription-factor residence time at the promoter (approximately 170 ms). These FRAP analysis tools will be important for measuring key cellular binding parameters necessary for a complete and accurate description of the networks that regulate cellular behavior.

Tumor cells depend on both glycolysis and oxidative phosphorylation (OXPHOS) for energy and biomass production to support cell proliferation. Recent data has demonstrated a dependence of various tumor types on mitochondrial OXPHOS, which represents an exciting therapeutic opportunity. Through an extensive medicinal chemistry campaign, IACS-010759 was identified as a potent, selective inhibitor of complex I of the electron transport chain, which is orally bioavailable and has excellent PK and physicochemical properties in preclinical species. Our group and others have demonstrated that AML, plus subsets of glioblastoma, neuroblastoma, lymphoma, melanoma, triple negative breast cancer (TNBC) and pancreatic cancer (PDAC) are highly dependent on OXPHOS to meet energy and biomass demands. Treatment of multiple cell lines and patient derived xenograft (PDX) models in several cancer types with IACS-010759 led to a robust decrease in cell viability and often an increase in apoptosis with EC50 values between 1 nM - 50 nM across multiple lines. Through a series of mechanistic studies we established that IACS-10759 blocks complex I of the electron transport at the quinone binding site. Mechanistically, response to IACS-010759 was associated with induction of a metabolic imbalances that negatively impacted energy homeostasis, aspartate biosynthesis, and NTP production due to reduced conversion of NADH to NAD+ by complex I, decreased ATP production, TCA cycle flux and nucleotide biosynthesis. Tumor growth inhibition and regression have been observed in molecularly defined subsets of TNBC and PDAC PDX xenograft models treated with IACS-010759, indicating that subsets of these indications are dependent on OXPHOS. Furthermore, treating TNBC or PDAC PDX models post-chemotherapy with IACS-010759 extends progression free survival, consistent with IACS-010759 targeting recently described metabolically adapted residual tumor cells. In orthotopic xenograft models of primary AML cells, daily oral treatment with 1-7.5 mg/kg IACS-010759 extended the median survival. Efficacy was paralleled by robust modulation of OCR, aspartate, and a gene signature levels. Therefore, these readouts (OCR, aspartate and a nanostring geneset) have been validated for use as exploratory clinical biology of response endpoints. In parallel, completion of preclinical chemistry, manufacturing and control (CMC) as well as GLP safety and tolerability studies with IACS-010759 in multiple species have enabled the selection of a clinical entry dose. As a result of the robust response in multiple cell lines, primary patient samples, and efficacy in PDX models, a Phase I clinical trial in relapsed, refractory AML was initiated in October 2016, with a parallel trial in solid tumors expected to initiate in early 2017. Initial results from the on-going AML trial will be disclosed. Citation Format: Jennifer Molina, Madhavi Bandi, Jennifer Bardenhagen, Christopher Bristow, Christopher Carroll, Edward Chang, Jason Cross, Naval Daver, Ningping Feng, Jason Gay, Mary Geck Do, Jennifer Greer, Jing Han, Judy Hirst, Sha Huang, Yongying Jiang, Zhijun Kang, Marina Konopleva, Gang Liu, Helen Ma, Polina Matre, Timothy McAfoos, Funda Meric-Bernstam, Pietro Morlacchi, Florian Muller, Marina Protopopova, Melinda Smith, Sonal Sonal, Yuting Sun, Jay Theroff, Andrea Viale, Quanyun Xu, Carlo Toniatti, Giulio Draetta, Philip Jones, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-010759, a novel inhibitor of complex I in Phase I clinical development to target OXPHOS dependent tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4971. doi:10.1158/1538-7445.AM2017-4971

Molecules targeting pro-inflammatory pathways have demonstrated beneficial effects in cancer treatment. More recently, combination of natural and synthetic anti-inflammatory drugs was suggested as an appealing strategy to inhibit tumor growth. Herein, we show that curcumin, a polyphenol from Curcuma longa and celecoxib induce apoptosis in hematopoietic cancer cell lines (Hel, Jurkat, K562, Raji, and U937). Further investigations on the most sensitive cell line, U937, indicated that these effects were tightly associated with an accumulation of the cells in S and G2/M for curcumin and in G0/G1 phase of cell cycle for celecoxib, respectively. The effect of celecoxib on cell cycle is associated with an induction of p27 and the down-regulation of cyclin D1. However, in the case of combination experiments, the pretreatment of U937 cells with celecoxib at non-apoptogenic concentrations counteracted curcumin-induced apoptosis. We found that this effect correlated with the prevention of the accumulation in S and G2/M phase of cell cycle induced by curcumin. Similar results have been obtained when celecoxib and curcumin were co-administrated at the same time. Overall our data suggest that this natural and synthetic drug combination is detrimental for cell death induction.

Tumor cells normally depend on both glycolysis and oxidative phosphorylation (OXPHOS) to provide the energy and macromolecule building blocks for rapid growth. Metabolic vulnerabilities caused by inactivation of glycolysis render tumor cells highly dependent on OXPHOS, and represent a therapeutic opportunity. Through an extensive medicinal chemistry campaign, we have identified IACS-10759 as a potent inhibitor of complex I of OXPHOS. IACS-10759 effectively inhibits ATP production and oxygen consumption in isolated mitochondria, and inhibits the conversion of NADH to NAD+ in immunoprecipitated complex I in low nM range. The exact subunit that IACS-10759 binds to is under investigation. Importantly, IACS-10759 is orally bioavailable with excellent physicochemical properties in preclinical species, and shows significant efficacy in multiple tumor indications both in vitro and in vivo. Specifically, in a glycolysis-deficient xenograft model, IACS-10759 causes robust tumor regression, but has no effect in the same model when glycolysis is restored. In addition, in AML where tumor cells have been shown to be highly OXPHOS-dependent, IACS-10759 robustly suppresses cell growth and induces apoptosis in both primary AML samples and cell lines in vitro, but not in normal patient-derived bone marrow cells. Significantly, IACS-10759 extends median survival by over 50 days in an AML orthotopic xenograft model. Furthermore, IACS-10759 also shows selective efficacy in other cell line panels including pancreatic cancer, non-small cell lung cancer and colorectal cancer, and has synergism with glycolysis inhibitors. In light of these results, we are currently performing IND enabling studies for IACS-10759, with first-in-human studies targeted for fourth quarter of 2015. Citation Format: Marina Protopopova, Madhavi Bandi, Yuting Sun, 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, Melinda Smith, Jay Theroff, Jing Han, Yuanqing Wu, Lynda Chin, Giulio Draetta, Philip Jones, Carlo Toniatti, M. Emilia Di Francesco, Joseph R. Marszalek. IACS-10759: A novel OXPHOS inhibitor that selectively kills tumors with metabolic vulnerabilities. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A65.

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

Cardiac glycosides (CGs), prescribed to treat cardiovascular alterations, display potent anti-cancer activities. Despite their well-established target, the sodium/potassium (Na+/K+)-ATPase, downstream mechanisms remain poorly elucidated. UNBS1450 is a hemi-synthetic cardenolide derived from 2″-oxovorusharin extracted from the plant Calotropis procera, which is effective against various cancer cell types with an excellent differential toxicity. By comparing adherent and non-adherent cancer cell types, we validated Mcl-1 as a general and early target of UNBS1450. A panel of CGs including cardenolides ouabain, digitoxin and digoxin as well as bufadienolides cinobufagin and proscillaridin A allowed us to generalize our findings. Our results show that Mcl-1, but not Bcl-xL nor Bcl-2, is rapidly downregulated prior to induction of apoptosis. From a mechanistic point of view, we exclude an effect on transcription and demonstrate involvement of a pathway affecting protein stability and requiring the proteasome in the early CG-induced Mcl-1 downregulation, without the involvement of caspases or the BH3-only protein NOXA. Strategies aiming at preventing UNBS1450-induced Mcl-1 downregulation by overexpression of a mutated, non-ubiquitinable form of the protein or the use of the proteasome inhibitor MG132 inhibited the compound’s ability to induce apoptosis. Altogether our results point at Mcl-1 as a ubiquitous factor, downregulated by CGs, whose modulation is essential to achieve cell death.