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Acute myeloid leukemia (AML) is driven by numerous molecular events that contribute to disease progression. Herein, we identify hnRNP K overexpression as a recurrent abnormality in AML that negatively correlates with patient survival. Overexpression of hnRNP K in murine fetal liver cells results in altered self-renewal and differentiation potential. Further, murine transplantation models reveal that hnRNP K overexpression results in myeloproliferation in vivo. Mechanistic studies expose a direct functional relationship between hnRNP K and RUNX1—a master transcriptional regulator of hematopoiesis often dysregulated in leukemia. Molecular analyses show that overexpression of hnRNP K results in an enrichment of an alternatively spliced isoform of RUNX1 lacking exon 4. Our work establishes hnRNP K’s oncogenic potential in influencing myelogenesis through its regulation of RUNX1 splicing and subsequent transcriptional activity.

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.

We and others have reported that the anticancer activity of L-asparaginase (ASNase) against asparagine synthetase (ASNS)-positive cell types requires ASNase glutaminase activity, whereas anticancer activity against ASNS-negative cell types does not. Here, we attempted to disentangle the relationship between asparagine metabolism, glutamine metabolism, and downstream pathways that modulate cell viability by testing the hypothesis that ASNase anticancer activity is based on asparagine depletion rather than glutamine depletion per se. We tested ASNase wild-type (ASNaseWT) and its glutaminase-deficient Q59L mutant (ASNaseQ59L) and found that ASNase glutaminase activity contributed to durable anticancer activity against xenografts of the ASNS-negative Sup-B15 leukemia cell line in NOD/SCID gamma mice, whereas asparaginase activity alone yielded a mere growth delay. Our findings suggest that ASNase glutaminase activity is necessary for durable, single-agent anticancer activity in vivo, even against ASNS-negative cancer types.

Background Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is an RNA-binding protein that is aberrantly expressed in cancers. We and others have previously shown that reduced hnRNP K expression downmodulates tumor-suppressive programs. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon, yet its functional consequences and clinical significance remain unknown. Methods Clinical implications of hnRNP K overexpression were examined through immunohistochemistry on samples from patients with diffuse large B-cell lymphoma who did not harbor MYC alterations (n = 75). A novel transgenic mouse model that overexpresses hnRNP K specifically in B cells was generated to directly examine the role of hnRNP K overexpression in mice (three transgenic lines). Molecular consequences of hnRNP K overexpression were determined through proteomics, formaldehyde-RNA-immunoprecipitation sequencing, and biochemical assays. Therapeutic response to BET-bromodomain inhibition in the context of hnRNP K overexpression was evaluated in vitro and in vivo (n = 3 per group). All statistical tests were two-sided. Results hnRNP K is overexpressed in diffuse large B-cell lymphoma patients without MYC genomic alterations. This overexpression is associated with dismal overall survival and progression-free survival (P Conclusion Our findings indicate that hnRNP K is a bona fide oncogene when overexpressed and represents a novel mechanism for c-Myc activation in the absence of MYC lesions.

SummaryAcute myeloid leukemia (AML) is driven by numerous molecular events that contribute to disease progression. Herein, we identified hnRNP K overexpression as a recurrent abnormality in AML that is associated with inferior patient outcomes. In murine hematopoietic stem and progenitor cells, hnRNP K overexpression altered self-renewal and differentiation potential. Furthermore, murine transplantation models revealed that hnRNP K overexpression resulted in fatal myeloproliferative phenotypes. Using unbiased approaches, we discovered a direct relationship between hnRNP K and RUNX1—a master transcriptional regulator of hematopoiesis often dysregulated in leukemia. Molecular analyses revealed hnRNP K-dependent alternative splicing of RUNX1, resulting in the generation of a functionally distinct isoform. Taken together, we have established hnRNP K as an oncogene in myeloid leukemia that binds RUNX1 RNA, altering its splicing and subsequent transcriptional activity. These findings shed new light on a mechanism of myeloid leukemogenesis, paving the way for new drug discovery efforts.HighlightshnRNP K, an RNA binding protein, is overexpressed in AML and correlates with poor clinical outcomeshnRNP K overexpression in murine HSPCs drives fatal myeloproliferative phenotypes in micehnRNP K’s oncogenicity can be attributed, at least in part, to its ability to bind and influence the splicing of the RUNX1 transcript

We present a Chemistry and Structure Screen Integrated Efficiently (CASSIE) approach (named for Greek prophet Cassandra) to design inhibitors for cancer biology and pathogenesis. CASSIE provides an effective path to target master keys to control the repair-replication interface for cancer cells and SARS CoV-2 pathogenesis as exemplified here by specific targeting of Poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribose glycohydrolase ARH3 macrodomains plus SARS CoV-2 nonstructural protein 3 (Nsp3) Macrodomain 1 (Mac1) and Nsp15 nuclease. As opposed to the classical massive effort employing libraries with large numbers of compounds against single proteins, we make inhibitor design for multiple targets efficient. Our compact, chemically diverse, 5000 compound Goldilocks (GL) library has an intermediate number of compounds sized between fragments and drugs with predicted favorable ADME (absorption, distribution, metabolism, and excretion) and toxicological profiles. Amalgamating our core GL library with an approved drug (AD) library, we employ a combined GLAD library virtual screen, enabling an effective and efficient design cycle of ranked computer docking, top hit biophysical and cell validations, and defined bound structures using human proteins or their avatars. As new drug design is increasingly pathway directed as well as molecular and mechanism based, our CASSIE approach facilitates testing multiple related targets by efficiently turning a set of interacting drug discovery problems into a tractable medicinal chemistry engineering problem of optimizing affinity and ADME properties based upon early co-crystal structures. Optimization efforts are made efficient by a computationally-focused iterative chemistry and structure screen. Thus, we herein describe and apply CASSIE to define prototypic, specific inhibitors for PARG vs distinct inhibitors for the related macrodomains of ARH3 and SARS CoV-2 Nsp3 plus the SARS CoV-2 Nsp15 RNA nuclease.

Arrival of the novel SARS-CoV-2 has launched a worldwide effort to identify both pre-approved and novel therapeutics targeting the viral proteome, highlighting the urgent need for efficient drug discovery strategies. Even with effective vaccines, infection is possible, and at-risk populations would benefit from effective drug compounds that reduce the lethality and lasting damage of COVID-19 infection. The CoV-2 MacroD-like macrodomain (Mac1) is implicated in viral pathogenicity by disrupting host innate immunity through its mono (ADP-ribosyl) hydrolase activity, making it a prime target for antiviral therapy. We therefore solved the structure of CoV-2 Mac1 from non-structural protein 3 (Nsp3) and applied structural and sequence-based genetic tracing, including newly determined A. pompejana MacroD2 and GDAP2 amino acid sequences, to compare and contrast CoV-2 Mac1 with the functionally related human DNA-damage signaling factor poly (ADP-ribose) glycohydrolase (PARG). Previously, identified targetable features of the PARG active site allowed us to develop a pharmacologically useful PARG inhibitor (PARGi). Here, we developed a focused chemical library and determined 6 novel PARGi X-ray crystal structures for comparative analysis. We applied this knowledge to discovery of CoV-2 Mac1 inhibitors by combining computation and structural analysis to identify PARGi fragments with potential to bind the distal-ribose and adenosyl pockets of the CoV-2 Mac1 active site. Scaffold development of these PARGi fragments has yielded two novel compounds, PARG-345 and PARG-329, that crystallize within the Mac1 active site, providing critical structure-activity data and a pathway for inhibitor optimization. The reported structural findings demonstrate ways to harness our PARGi synthesis and characterization pipeline to develop CoV-2 Mac1 inhibitors targeting the ADP-ribose active site. Together, these structural and computational analyses reveal a path for accelerating development of antiviral therapeutics from pre-existing drug optimization pipelines.

Mutations in RNA binding proteins have been identified as pathogenic drivers in many hematological malignancies. However, in addition to mutational status, expression changes in RNA binding proteins likely impact disease processes. Through our studies, we identified that overexpression of hnRNP K (heterogeneous ribonucleoprotein K) -a poly(C)-RNA binding protein that governs the expression of numerous genes and transcripts- plays a pivotal role in myeloid malignancies. Using clinical samples, we determined that hnRNP K overexpression is a recurrent abnormality, occurring in nearly 30% of AML cases. Importantly, elevated hnRNP K levels associate with decreased overall survival (24.3 months versus 48.7 months; HR 1.9; 95% CI 1.3-2.7). However, the role of hnRNP K overexpression in AML remains unclear. To evaluate its putative oncogenic potential, we overexpressed hnRNP K in murine fetal liver cells (FLCs). Using colony formation assays (CFAs), we demonstrated that hnRNP K-overexpressing FLCs have an altered differentiation potential (increased number of immature (c-kit +Sca-1 +) and decreased number of mature myeloid (Gr1 +CD11b +) cells) and an increase in self-renewal capacity (increased number of colonies) (p=0.008). Mice transplanted with hnRNP K overexpressing FLCs had markedly shortened survival compared to empty vector controls, despite similar engraftment (median survival 8.1 weeks versus median not reached (HR 3.0, 95% CI 1.2 - 7.3, p=0.02). Significantly, extramedullary hematopoiesis was observed in the spleens and the hepatic parenchyma of mice transplanted with FLCs that overexpress hnRNP K. This resulted in disrupted splenic architecture and the presence of immature hematopoietic cells and cells of myeloid origin (CD117, CD14, and myeloperoxidase). Furthermore, analyses of the bone marrow revealed an increase in myeloid cells in hnRNP K transplanted mice. We next used unbiased and biochemical approaches to discover a direct interaction between hnRNP K and the RUNX1 transcript-a critical transcriptional factor often dysregulated in leukemia. Molecular analyses revealed hnRNP K-dependent alternative splicing of RUNX1 (delExon6) , resulting in the generation of a functionally distinct isoform that is more stable than full-length RUNX1. RNA-Seq and reporter assays demonstrated that delExon6 has a unique transcriptional profile compared to full-length RUNX1, suggesting this spliced transcript may have a pathogenic role. To examine the functionality of delExon6, we performed CFAs. Here, we observed that delExon6 expression results in an increased proliferation potential that is mediated by hnRNP K's RNA binding activity. Together, these data establish hnRNP K as an oncogene in myeloid leukemia through its ability to directly bind the RUNX1 transcript, modify RUNX1 splicing, and subsequently alter its transcriptional activity. Disclosures No relevant conflicts of interest to declare.

hnRNP K (heterogeneous ribonucleoprotein K) is an RNA-binding protein that binds to conserved poly-C rich tracks in RNA and influences a diverse set of molecular pathways involved in tumorigenesis. Our previous studies identified hnRNP K overexpression in patients with diffuse large B-cell lymphoma (46/75, 61%) and acute myeloid leukemia (45/160, 28%). This overexpression correlates with dismal clinical outcomes and a lack of therapeutic responses to standard treatment. To explore hnRNP K's in vivo functions, we generated Hnrnpk-transgenic mouse models. These mice develop lymphoma phenotypes through activation of the c-Myc pathway. In pre-clinical settings, bromodomain inhibitors disrupted hnRNP K-mediated c-Myc activation, demonstrating that hnRNP K overexpression mediated-pathways are amenable to therapeutic intervention. To further our studies, we used IP-mass spectrometry, RNA-sequencing, RNA immunoprecipitation, reverse phase protein analyses, and polysome profiling to identify novel pathways associated with changes in hnRNP K expression. Here, we observed that alterations in hnRNP K expression result in an impairment of ribosomal biogenesis and activation of pathways directly responsible for global translation. Using both knockdown and overexpression systems, we observed a direct correlation between hnRNP K expression and expression of S6, S6K, phosphorylated S6, eIF and mTOR pathways and uncovered defects in rRNA splicing. Collectively, these data indicate that impairment of cap-dependent loading and alterations in ribogenesis may be a driving force in the clinical manifestations of hnRNP K-driven malignancies. Furthermore, these results suggest that translational-inhibitors may be useful in exploiting hnRNP K-dependent vulnerabilities. To examine this aspect, we are currently using FDA-approved translation inhibitors and disruptors of ribogenesis (e.g. homoharringtonineand mTOR-inhibitors) and KTP- compounds, respectively. While these indirect targeting strategies are interesting, our results indicate that hnRNP K also regulates cellular programs outside of translation. Thus, potential therapies that effectively target hnRNP K overexpression will require direct inhibition of its RNA binding functions. To this end, we used several screening assays including fluorescence anisotropy (FA), surface plasmon resonance, SYPRO-orange thermal shift assays, and cell proliferation assays to screen 80,000 small molecule compounds which led to the identification of 9 candidates that disrupt hnRNP K-mRNA interactions and cause cell death in an hnRNP K-dependent manner. Further, cellular thermal shift assays revealed these lead compounds engage hnRNP K within cells and most critically, result in reduced expression of hnRNP K targets in vivo. These candidate compounds as well as potentially more potent structural analogs are currently being evaluated. Collectively, our results demonstrate that the oncogenic functions of hnRNP K are amenable to both indirect therapeutic intervention using FDA-approved agents as well as direct inhibition through newly identified small molecule compounds, signifying that there may be a roadmap to effective therapies for hnRNP K-dependent malignancies. Disclosures No relevant conflicts of interest to declare.

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization, thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel x-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

We recently reported that SF2312 ((1,5-dihydroxy-2-oxopyrrolidin-3-yl)phosphonic acid), a phosphonate antibiotic with a previously unknown mode of action, is a potent inhibitor of the glycolytic enzyme, Enolase. SF2312 can only be synthesized as a racemic-diastereomeric mixture. However, co-crystal structures with Enolase 2 (ENO2) have consistently shown that only the (3S,5S)-enantiomer binds to the active site. The acidity of the alpha proton at C-3, which deprotonates under mildly alkaline conditions, results in racemization; thus while the separation of four enantiomeric intermediates was achieved via chiral High Performance Liquid Chromatography (HPLC) of the fully protected intermediate, deprotection inevitably nullified enantiopurity. To prevent epimerization of the C-3, we designed and synthesized MethylSF2312, ((1,5-dihydroxy-3-methyl-2-oxopyrrolidin-3-yl)phosphonic acid), which contains a fully-substituted C-3 alpha carbon. As a racemic-diastereomeric mixture, MethylSF2312 is equipotent to SF2312 in enzymatic and cellular systems against Enolase. Chiral HPLC separation of a protected MethylSF2312 precursor resulted in the efficient separation of the four enantiomers. After deprotection and inevitable re-equilibration of the anomeric C-5, (3S)-MethylSF2312 was up to 2000-fold more potent than (3R)-MethylSF2312 in an isolated enzymatic assay. This observation strongly correlates with biological activity in both human cancer cells and bacteria for the 3S enantiomer of SF2312. Novel X-ray structures of human ENO2 with chiral and racemic MethylSF2312 show that only (3S,5S)-enantiomer occupies the active site. Enolase inhibition is thus a direct result of binding by the (3S,5S)-enantiomer of MethylSF2312. Concurrent with these results for MethylSF2312, we contend that the (3S,5S)-SF2312 is the single active enantiomer of inhibitor SF2312.

Despite being crucial for energy generation in most forms of life, few if any microbial antibiotics specifically inhibit glycolysis. To develop a specific inhibitor of the glycolytic enzyme enolase 2 (ENO2) for the treatment of cancers with deletion of ENO1 (encoding enolase 1), we modeled the synthetic tool compound inhibitor phosphonoacetohydroxamate (PhAH) into the active site of human ENO2. A ring-stabilized analog of PhAH, in which the hydroxamic nitrogen is linked to Cα by an ethylene bridge, was predicted to increase binding affinity by stabilizing the inhibitor in a bound conformation. Unexpectedly, a structure-based search revealed that our hypothesized backbone-stabilized PhAH bears strong similarity to SF2312, a phosphonate antibiotic of unknown mode of action produced by the actinomycete Micromonospora, which is active under anaerobic conditions. Here, we present multiple lines of evidence, including a novel X-ray structure, that SF2312 is a highly potent, low-nanomolar inhibitor of enolase.

Summary The mitochondrial caseinolytic protease P (ClpP) plays a central role in mitochondrial protein quality control by degrading misfolded proteins. Using genetic and chemical approaches, we showed that hyperactivation of the protease selectively kills cancer cells, independently of p53 status, by selective degradation of its respiratory chain protein substrates and disrupts mitochondrial structure and function, while it does not affect non-malignant cells. We identified imipridones as potent activators of ClpP. Through biochemical studies and crystallography, we show that imipridones bind ClpP non-covalently and induce proteolysis by diverse structural changes. Imipridones are presently in clinical trials. Our findings suggest a general concept of inducing cancer cell lethality through activation of mitochondrial proteolysis.

ClpP is a mitochondrial protease and a major protein quality control mediator that primarily interacts with metabolic enzymes in mitochondria. Here, we demonstrate that activation of this protease results in prominent anti-cancer activity, and propose ClpP activation as a novel therapeutic strategy for cancer and hematologic malignancies. We used genetic and chemical tools to activate ClpP. In a genetic approach, we tested the anti-cancer effects of ClpP activation by expressing a constitutively active ClpP mutant. Indeed, induction of the active ClpP mutant induced apoptosis in vitro and inhibited tumor progression in vivo. To further explore the antineoplastic effects of ClpP activation, we then performed a chemical screen of an in-house library of on-patent and off-patent drugs and identified imipridones (ONC201 and ONC212) as potent ClpP agonists. Imipridones are first-in-class antineoplastic agents and have shown preclinical efficacy in various malignancies in vitro and in vivo and are currently being evaluated in clinical trials in a diverse spectrum of cancers. Importantly, we and others have shown that their activity is agnostic to TP53 mutational status. Of note, molecular targets of imipridones that bind the drugs and are functionally important for their cytotoxicity have never been identified. Through extensive chemical investigations, including analysis of binding mechanism of the compounds to ClpP in cell free (ITC) and cell based assays (CETSA) as well as molecular analysis of the crystal structure, we demonstrate that these molecules bind ClpP non-covalently, and activate the protease by stabilizing the ClpP 14-mer, enlarging the axial pores of the complex, and inducing structural changes in the residues surrounding and including the catalytic triad. In leukemia, lymphoma and colon cancer cells including primary acute myeloid leukemia (AML) cells, both compounds displayed potent ClpP-dependent cytotoxicity with IC50s in low micro- or nanomolar ranges. Importantly, in primary AML samples, pretreatment ClpP levels correlated with response to imipridones. In lymphoma and AML xenograft models, both genetic and chemical activation of ClpP resulted in antitumor effects, while expression of inactive D190A ClpP mutant induced resistance. Mechanistically, ClpP activation leads to increased degradation of substrates of the enzyme including respiratory chain complex subunits and mitochondrial translation system. The resultant impaired mitochondrial structure and reduction in oxygen consumption is selectively cytotoxic to malignant cells that rely highly on mitochondrial energy production for their survival, whereas normal cells are not affected. In conclusion, ClpP activation is an entirely novel therapeutic strategy for malignant tumors. Our findings also suggest a general concept of inducing TP53-independent cancer cell lethality through activation of mitochondrial proteolysis. Citation Format: Jo Ishizawa, Sarah F. Zarabi, R Eric Davis, Ondrej Halgas, Takenobu Nii, Yulia Jitkova, Ran Zhao, Jonathan St-Germain, Lauren E. Heese, Grace Egan, Vivian R. Ruvolo, Samir H. Barghout, Yuki Nishida, Rose Hurren, Wencai Ma, Marcela Gronda, Todd Link, Keith Wong, Mark Mabanglo, Kensuke Kojima, Gautam Borthakur, Neil MacLean, John Man Chun Ma, Andrew B. Leber, Mark D. Minden, Walid Houry, Hagop Kantarjian, Martin Stogniew, Brian Raught, Emil F. Pai, Aaron D. Schimmer, Michael Andreeff. Mitochondrial ClpP-mediated proteolysis induces selective cancer cell lethality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2720.

Due to the rolling manufacturing process, most advanced high-strength steels (AHSS) demonstrate in-plane anisotropic material behavior. This study investigates the effects of material orientation on the axial crush behavior and fracture of AHSS with axial crush tests and computer simulations.Crush simulation models considering material anisotropy and damage evolution were developed in LS-DYNA based on the drop-tower crush test results and coupon characterization test data for DP780 steel. The modified Mohr-Coulomb (MMC) isotropic fracture model was employed in the crush simulation models for fracture prediction.The 12-sided components fabricated in the transverse (T) direction of the sheet exhibited slightly higher crush loads and reduced crush distances compared to those in the longitudinal (L) direction. The crush behavior in each direction was generally proportional to ultimate tensile strength. All of the materials investigated in this study showed some cracking in the crush tests for both component orientations, but only DP780 showed significant anisotropy in fracture behavior with more cracking for the T direction compared to the L direction. Overall, the amount of cracking observed in the tests had little or no significant effect on the axial crush performance. The MMC fracture loci in both the L and T directions were determined using a reverse engineering approach, and the stress-strain curves beyond the uniform elongation point were extended using an optimization method. Both material models MAT103 and MAT224 predicted the crush and fracture behavior with reasonably good accuracy.The predicted fracture mode and force-displacement curves agreed well with the test data for both the L and T directions in axial crush tests of the 12-sided components. The simple isotropic material model MAT224 is adequate for crush simulations to predict material orientation effects on AHSS component crush performance and fracture behavior.

© 2016, American Society for Biochemistry and Molecular Biology Inc. All rights reserved. Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKGII binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.

Summary HipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding "P loop motif." How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable "in-out" conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the "out state," which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host.

Acute myeloid leukemia (AML) is a conglomerate of hematologic malignancies characterized by recurrent genetic and/or chromosomal aberrations. Recent development of targeted agents has bolstered our armamentarium of therapeutic options and improved outcomes for many patients. Acquiring deeper mechanistic understanding of myeloid leukemogenesis will provide a basis for development of even more therapeutic strategies and further improve patient outcomes. Our clinical data have revealed that overexpression of HNRNPK is a recurrent abnormality that occurs in upwards of 20% of AML cases at both the RNA and protein levels. Using bone marrow samples from a similar proportion of patients, we have recently discovered a supernumerary marker chromosome containing an extra copy of the HNRNPK locus that is not detectable with routine cytogenetic testing. We have further associated high hnRNP K protein levels with decreased overall survival in de novo AML, emphasizing the need to understand the role of hnRNP K in myeloid malignancy. To directly evaluate the oncogenic capacity of hnRNP K, we have overexpressed hnRNP K in murine fetal liver cells (FLCs). Using CyTOF and colony formation assays, we demonstrated that hnRNP K-overexpressing FLCs have altered differentiation potential and self-renewal capacity compared to empty vector controls in vitro. These findings are recapitulated in vivo, as murine recipients of hnRNP K-overexpressing FLCs develop myeloid lineage disease, often manifesting as fatal megakaryocytic leukemia. To elucidate a mechanism by which hnRNP K causes myeloid disease, we performed hnRNP K immunoprecipitation followed by mass spectrometry in an AML cell line and identified that hnRNP K preferentially interacts with translational machinery, ribosomal subunits, and proteins involved in RNA processing. In conjunction with data from our hnRNP K overexpression models that indicate overexpression of hnRNP K occurs primarily in the cytoplasm, we then performed hnRNP K-RNA immunoprecipitation followed by sequencing (RIP-Seq). We determined that hnRNP K interacts with the transcript of RUNX1-a master regulator of hematopoiesis and a critical player in a myriad of leukemias-including megakaryocytic leukemias like those observed in our mouse models. Using biochemical assays, we have demonstrated that hnRNP K directly binds to consensus sequences in the RUNX1 transcript, and ultimately alters RUNX1 translation. Indeed, mice exhibiting hnRNP K overexpression have increased protein levels of Runx1 in hematopoietic tissues. Our data demonstrate that hnRNP K overexpression drives myeloid malignancy. Currently, we are screening compounds that will disrupt the interaction between hnRNP K and RUNX1 in our efforts to further understand myeloid biology and ultimately improve outcomes for patients with these diseases. Disclosures No relevant conflicts of interest to declare.

hnRNP K is an RNA binding protein that controls a multitude of cellular processes and is aberrantly expressed in cancers. We have previously shown that hnRNP K functions as a haploinsufficient tumor suppressor in AML patients with 9q deletions. However, overexpression of hnRNP K is the more commonly observed clinical phenomenon. We have recently discovered that hnRNP K overexpression in patients with diffuse large B-cell lymphoma correlates with dismal outcomes and directly resulted in the development of lymphomas in transgenic mice. To understand the mechanistic basis for the oncogenicity of hnRNP K overexpression and to identify therapeutic vulnerabilities, we performed RNA-sequencing, RNA immunoprecipitation following by sequencing (RIP-Seq), mass spectrometry, and polysome assays. We observed that hnRNP K regulates both global transcription and translational processes within the cell via modulation of 7SK and translation initiation proteins (such as the eIFs and PABP), respectively. Consequently, we hypothesized that aberrant hnRNP K expression primarily perturbs oncogenes with short half-lives. Mechanistically, we identified that hnRNP K binds to the RNA and regulates the expression of a plethora of critical oncogenes and tumor suppressors involved in hematologic malignancies such as c-Myc, RUNX1, and Cyclin D1. As proof of concept for clinical applications, we have demonstrated that hnRNP K-driven c-Myc overexpression renders tumors susceptible to bromodomain inhibition. Given that hnRNP K directs global transcription and translation, it is likely that hnRNP K overexpressing tumors will also be sensitive to transcriptional and translational inhibitors such as CDK9 inhibitors and omacetaxine mepesuccinate, respectively. However, since hnRNP K also regulates a plethora of additional cellular processes that extend far beyond mRNA and protein synthesis, there is a need to develop hnRNP K-specific inhibitors that will only target these activities. Thus, we have recently begun to identify small molecule compounds that can directly inhibit hnRNP K-RNA binding function on specific targets using an in vitro fluorescent binding assay. Using this assay, we are currently screening a library of 70,000 small molecule compounds to identify agents that can prevent hnRNP K-RNA interactions. In summary, we have established that hnRNP K is a bona fide oncogene that drives lymphomagenesis. Global analyses have revealed therapeutic vulnerabilities of hnRNP K overexpressing tumors. Furthermore, using our in vitro RNA binding assays, we anticipate identification of novel hnRNP K-specific inhibitors. Disclosures No relevant conflicts of interest to declare.

Homeodomain (HD) transcriptional activities are tightly regulated during embryogenesis and require protein interactions for their spatial and temporal activation. The chromatin-associated high mobility group protein (HMG-17) is associated with transcriptionally active chromatin, however its role in regulating gene expression is unclear. This report reveals a unique strategy in which, HMG-17 acts as a molecular switch regulating HD transcriptional activity. The switch utilizes the Wnt/beta-catenin signaling pathway and adds to the diverse functions of beta-catenin. A high-affinity HMG-17 interaction with the PITX2 HD protein inhibits PITX2 DNA-binding activity. The HMG-17/PITX2 inactive complex is concentrated to specific nuclear regions primed for active transcription. beta-Catenin forms a ternary complex with PITX2/HMG-17 to switch it from a repressor to an activator complex. Without beta-catenin, HMG-17 can physically remove PITX2 from DNA to inhibit its transcriptional activity. The PITX2/HMG-17 regulatory complex acts independently of promoter targets and is a general mechanism for the control of HD transcriptional activity. HMG-17 is developmentally regulated and its unique role during embryogenesis is revealed by the early embryonic lethality of HMG-17 homozygous mice. This mechanism provides a new role for canonical Wnt/beta-catenin signaling in regulating HD transcriptional activity during development using HMG-17 as a molecular switch.

Recent studies on Hfq have provided a deeper understanding of the multiple functions of this pleiotropic post-transcriptional regulator. Insights into the mechanism of Hfq action have come from a variety of approaches. A key finding was the characterization of two RNA binding sites: the Proximal Site, which binds sRNA and mRNA; and the Distal Site, which binds poly(A) tails. Hfq was shown to interact with PAP I, PNP and RNase E, proteins that are involved in mRNA decay and in vitro, was shown to form fibres, the physiological significance of which is unknown. Fluorescence resonance energy transfer (FRET) studies directly demonstrated the role of Hfq as a chaperone that facilitates the interaction between sRNAs and target mRNAs. There are still, however, some unresolved questions.

Signal transducer and activator of transcription 6 (STAT6) transmits signals from cytokines IL-4 and IL-13 and is activated in allergic airway disease. We are developing phosphopeptide mimetics targeting the SH2 domain of STAT6 to block recruitment to phosphotyrosine residues on IL-4 or IL-13 receptors and subsequent Tyr641 phosphorylation to inhibit the expression of genes contributing to asthma. Structure-affinity relationship studies showed that phosphopeptides based on Tyr631 from IL-4Rα bind with weak affinity to STAT6, whereas replacing the pY+3 residue with simple aryl and alkyl amides resulted in affinities in the mid to low nM range. A set of phosphatase-stable, cell-permeable prodrug analogues inhibited cytokine-stimulated STAT6 phosphorylation in both Beas-2B human airway cells and primary mouse T-lymphocytes at concentrations as low as 100 nM. IL-13-stimulated expression of CCL26 (eotaxin-3) was inhibited in a dose-dependent manner, demonstrating that targeting the SH2 domain blocks both phosphorylation and transcriptional activity of STAT6.

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.

Glycolysis inhibition is an active area of investigation for the treatment of cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions solely reliant on ENO2, rendering them exquisitely sensitive to enolase inhibitors Collateral Lethality. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We recently reported that a structural analogue of PhAH, the natural phosphonate antibiotic SF2312, is a high potency inhibitor of Enolase. While more potent than PhAH, SF2312 remains poorly cell permeable. Here, we generated a Pivaloyloxymethyl (POM) ester pro-drug derivative of SF2312, termed POMSF, which increased the potency in cell based systems by ~50-fold. POMSF is selectively active against ENO1-deleted glioma cells in culture at ~19 nM, versus μM for SF2312. However, POMSF displayed poor aqueous stability. A derivative of POMSF, termed POMHEX, showed greater stability and its active form, HEX, showed 4-fold preference for ENO1 over ENO2. Labeled 13C-glucose tracing shows that POMHEX inhibits glycolysis at the Enolase step in all cell lines tested, but with ~100-fold greater potency in ENO1-deleted lines. POMHEX selectively killed ENO1-deleted glioma cells with an IC50 Citation Format: Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Federica Pisaneschi, Paul Leonard, David Maxwell, Zhenghong Peng, Todd Link, Lee IV R. Gilbert, Ananth Bosajou, Duoli Sun, Joe Marszalek, Yuting Sun, John S. McMurray, Pijus K. Mandal, Maria E. Di Francesco, Barbara Czako, Alan Wang, William Bornmann, Ronald A. DePinho, Florian Muller. Pomhex, a cell-permeable high potency enolase inhibitor with utility for collateral lethality treatment of cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A39.

Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5′-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.

Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.

(1994). MONOSUBSTITUTED 2,2′-BIPYRIDINES. Organic Preparations and Procedures International: Vol. 26, No. 6, pp. 696-701.

Glycolysis inhibition is an active area of investigation in cancer. However, few compounds have progressed beyond the cell culture stage. We have recently demonstrated that genomic passenger deletion of the glycolytic enzyme Enolase 1 (ENO1) leaves gliomas harboring such deletions with less than 10% of normal enzymatic activity, rendering them exquisitely sensitive to enolase inhibitors. However, the tool compound that we employed for these in vitro studies, Phosphonoacetohydroxamate (PhAH), has very poor pharmacological properties and was ineffective in vivo. We performed a SAR studies to increase inhibitor specificity towards ENO2 as well as pro-druging to increase cell permeability. The lead compound generated by these efforts, termed POMHEX, is selectively active against ENO1-deleted glioma cells in culture at ∼35nM (versus μM for PhAH). Using an orthotopic intracranial xenografted model where tumor growth and response to therapy are monitored by MRI, we show that POMHEX is capable of eradicating intracranial ENO1-deleted tumors, with mice remaining recurrence-free even after treatment discontinuation. Taken together, these results reinforce that glycolysis is a viable target and provide in vivo proof-of-principal for the concept of using passenger deletions as targetable vulnerabilities in cancer therapy. Citation Format: Yu-Hsi Lin, Joe Marszalek, Yuting Sun, Naima Hammoudi, Paul Leonard, David Maxwell, Nikunj Satani, Peng Zhang, Todd Link, Gilbert Lee, Maria E. Di Francesco, Barbara Czako, Alan Y. Want, Ronald A. DePinho, Florian L. Muller. Pomhex: a cell-permeable high potency Enolase inhibitor with in vivo anti-neoplastic activity. [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 C183.

(1994). MONOSUBSTITUTED 2,2′-BIPYRIDINES. Organic Preparations and Procedures International: Vol. 26, No. 6, pp. 696-701.

Hfq is a small, highly abundant hexameric protein that is found in many bacteria and plays a critical role in mRNA expression and RNA stability. As an “RNA chaperone,” Hfq binds AU-rich sequences and facilitates the trans annealing of small RNAs (sRNAs) to their target mRNAs, typically resulting in the down-regulation of gene expression. Hfq also plays a key role in bacterial RNA decay by binding tightly to polyadenylate [poly(A)] tracts. The structural mechanism by which Hfq recognizes and binds poly(A) is unknown. Here, we report the crystal structure of Escherichia coli Hfq bound to the poly(A) RNA, A 15 . The structure reveals a unique RNA binding mechanism. Unlike uridine-containing sequences, which bind to the “proximal” face, the poly(A) tract binds to the “distal” face of Hfq using 6 tripartite binding motifs. Each motif consists of an adenosine specificity site (A site), which is effected by peptide backbone hydrogen bonds, a purine nucleotide selectivity site (R site), and a sequence-nondiscriminating RNA entrance/exit site (E site). The resulting implication that Hfq can bind poly(A-R-N) triplets, where R is a purine nucleotide and N is any nucleotide, was confirmed by binding studies. Indeed, Hfq bound to the oligoribonucleotides (AGG) 8 , (AGC) 8 , and the shorter (A-R-N) 4 sequence, AACAACAAGAAG, with nanomolar affinities. The abundance of (A-R-N) 4 and (A-R-N) 5 triplet repeats in the E. coli genome suggests additional RNA targets for Hfq. Further, the structure provides insight into Hfq-mediated sRNA-mRNA annealing and the role of Hfq in RNA decay.

The influence of strain rate on work hardening behavior has been determined for a variety of high strength steels including high-strength low-alloy (HSLA), dual phase (DP), and transformation-induced plasticity (TRIP) steels. Tensile testing was performed at true strain rates of 10 - 3 s - 1 and 1.0 s - 1 to represent laboratory testing conditions and dynamic press-forming operations, respectively. Work hardening behavior is described by the conventional strain hardening exponent (n-value), the work hardening rate (dσ/de), and the Shape-Tilt-Strength (STS) equation as an alternative approach. The effects of deformation temperature and temperature rise during deformation (adiabatic heating) on work hardening are also evaluated. Increasing the strain rate generally increases the work hardening rate at smaller strains, which may contribute to a broader initial strain distribution in press forming. At larger strains, the work hardening rate decreases with increasing strain rate due to adiabatic heating, thereby lowering the overall forming limits. The high-strain-rate formability decrease due to adiabatic heating is anticipated to be greater as strength level increases for a given degree of formability.

Membrane bound type II cGMP dependent protein kinase (PKG II) is a central mediator of cGMP signaling cascade, which regulates circadian rhythmicity, intestinal water secretion, bone growth and renal functions. PKG II contains an N-terminal regulatory (R)-domain, and a C-terminal catalytic (C)-domain. The R-domain contains tandem cyclic nucleotide binding domains (CNB-A and B) each with different affinities for cGMP, the second messenger that regulates kinase activity of PKG II. While it is known that PKG II needs to be highly selective for cGMP over cAMP to function properly, little is known about its cyclic nucleotide selectivity and the selectivity's role in activation. To understand its cyclic nucleotide selectivity and activation mechanism of PKG II, we first identified CNB-B to be highly selective for cGMP and solved its crystal structure with cGMP. The complex structure revealed that PKG II utilizes an arginine and two aspartate residues on the C-terminal helix to recognize the guanine moiety in cGMP. This is completely different from PKG I, where a conserved arginine from the β barrel of CNB-B specifically binds the guanine moiety of cGMP and imparts cyclic nucleotide selectivity. We are currently testing the roles of PKG II specific interactions in cGMP selectivity and activation of PKG II.

The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease.