Autophagy as a therapeutic mechanism to kill drug-resistant cancer cells.

Herein we discuss multiple pre-clinical projects developed by our group that have been translated into patients at Massey Cancer Center. Our work has used multi-kinase inhibitors, for example, sorafenib, regorafenib and neratinib, and combined with additional agents, for example, histone deacetylase inhibitors, the thymidylate synthase inhibitor pemetrexed, and PDE5 inhibitors. In broad-brush terms, our experience has been that these drug combinations enhance signaling by ATM-AMPK-ULK-1 and decrease signaling from growth factor receptors and RAS proteins, thereby lowering the activities of the intracellular signaling kinase ERK1/2, AKT, mTOR and p70 S6K . This collectively results in reduced protein synthesis and the induction of an endoplasmic reticulum stress response alongside autophagosome formation and autophagic flux. The rupture of autolysosomes, releasing proteases such as cathepsin B into the cytosol results in the cleavage and activation of the toxic BH3 domain protein BID which cooperates with BAX, BAK and BIM to cause mitochondrial dysfunction, leading to the release of cytochrome c and AIF, which then execute the tumor cell. For each of our two-drug combinations, we then performed additional laboratory-based studies to define the development of evolutionary resistance mechanisms, with the long-term concept of performing new three-drug clinical trials to prolong therapeutic efficacy and disease control.

A novel combination of isovanillin, curcumin, and harmine (GZ17-6.02) enhances cell death and alters signaling in actinic keratoses cells when compared to individual components and two-component combinations.

Actinic keratosis is a pre-malignant skin disease caused by excessive exposure to ultraviolet light. The present studies further defined the biology of a novel combination of isovanillin, curcumin, and harmine in actinic keratosis cells in vitro . An oral formulation (GZ17-6.02) and topical preparation (GZ21T) comprised of the same fixed, stoichiometric ratio have been developed. Together, the three active ingredients killed actinic keratosis cells more effectively than any of its component parts as either individual agents or when combined in pairs. The three active ingredients caused greater levels of DNA damage than any of its component parts as either individual agents or when combined in pairs. As a single agent, compared to isolated components, GZ17-6.02/GZ21T caused significantly greater activation of PKR-like endoplasmic reticulum kinase, the AMP-dependent protein kinase, and ULK1 and significantly reduced the activities of mTORC1, AKT, and YAP. Knockdown of the autophagy-regulatory proteins ULK1, Beclin1, or ATG5 significantly reduced the lethality of GZ17-6.02/GZ21T alone. Expression of an activated mammalian target of rapamycin mutant suppressed autophagosome formation and autophagic flux and reduced tumor cell killing. Blockade of both autophagy and death receptor signaling abolished drug-induced actinic keratosis cell death. Our data demonstrate that the unique combination of isovanillin, curcumin, and harmine represents a novel therapeutic with the potential to treat actinic keratosis in a manner different from the individual components or pairs of the components.

Cellular responses after (neratinib plus pemetrexed) exposure in NSCLC cells.

We previously demonstrated that neratinib interacted with pemetrexed to kill non-small cell lung cancer (NSCLC) cells. From developing other drug combinations, we observed that several days following exposure, cells activated survival mechanisms to counteract drug toxicity. The present studies attempted to define mechanisms that evolve to reduce the efficacy of neratinib and pemetrexed. Neratinib and pemetrexed synergized to kill NSCLC cells expressing wild-type RAS proteins, mutant KRAS (G12S; Q61H; G12A and G12C) or mutant NRAS (Q61K) or mutant ERBB1 (L858R; L858R T790M and exon 19 deletion). Neratinib and pemetrexed interacted in a greater than additive fashion to kill after 24 h, and after a further 24 h culture in the absence of drugs. Mutant KRAS G12V was more cytoprotective than either activated MEK1 or activated AKT. Knockdown of mutant KRAS reduced drug combination killing at the 48 h timepoint. Despite culture for 24 h in the absence of the drugs, the expression and activities of ERBB1, ERBB2 and ERBB4 remained significantly lower as did the activities of mammalian target of rapamycin (mTOR) C1 and mTORC2. The drug combination reduced KRAS and NRAS levels for 24 h, however, in the absence of the drugs, RAS levels had normalized by 48 h. Expression of Beclin1 and ATG5 remained elevated and of MCL1 and BCL-XL lower. No evolutionary activations of survival signaling by ERBB3, c-KIT, c-MET or PDGFRß or in intracellular signaling pathways were observed. These findings argue against the development of 'early' resistance mechanisms after neratinib and pemetrexed exposure. Future studies will be required to understand how NSCLC cells become resistant to neratinib and pemetrexed.

Mechanisms of GZ17-6.02 resistance.

OBJECTIVES: The drug GZ17-6.02 is undergoing phase I in solid tumor patients (NCT03775525). The present studies initially determined the impact of prolonged exposure of colorectal tumors to GZ17-6.02, and to determine whether GZ17-6.02 enhanced the efficacy of an anti-PD1 antibody. Subsequently, studies defined the evolutionary resistance mechanisms in tumor cells previously exposed to GZ17-6.02. METHODS: IACUC-approved animal studies were performed. In cell immunoblotting, cell transfections and trypan blue death assays were performed. RESULTS: Prolonged exposure of colorectal tumors to GZ17-6.02 enhanced the efficacy of 5-fluorouracil and of an anti-PD1 antibody, significantly prolonging animal survival. Tumor cells previously exposed to GZ17-6.02 in vivo had elevated their expression of ERBB2 and ERBB3, and increased phosphorylation of ERBB1, ERBB3, PDGFRß, AKT T308, ERK1/2, p70 S6K T389, STAT5 Y694 and c-SRC Y416. The phosphorylation of c-SRC Y527 declined. The efficacy of ERBB receptor inhibitors at killing these resistant tumor cells was unaltered by prior GZ17-6.02 exposure whereas the efficacy of multi-kinase/PDGFRß inhibitors was significantly reduced. Treatment of colon cancer cells with GZ17-6.02 rapidly reduced the levels of multiple HDAC proteins and altered their subcellular localization. Isolates from resistant tumors expressed less CD95 and FAS-L. HDAC inhibitors enhanced CD95 and FAS-L levels in the resistant cells via activation of NFκB and HDAC inhibitors restored the efficacy of GZ17-6.02 to near control levels. CONCLUSIONS: Our findings demonstrate that GZ17-6.02 has the potential to be developed as a colon cancer therapeutic and that resistance to the drug can be partially reversed by HDAC inhibitors.

The role of cell signaling in the crosstalk between autophagy and apoptosis in the regulation of tumor cell survival in response to sorafenib and neratinib.

The molecular mechanisms by which tumor cells survive or die following therapeutic interventions are complex. There are three broadly defined categories of cell death processes: apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). In hematopoietic tumor cells, the majority of toxic stimuli cause these cells to undergo a death process called apoptosis; apoptosis specifically involves the cleavage of DNA into large defined pieces and their subsequent localization in vesicles. Thus, 'pure' apoptosis largely lacks inflammatory potential. In carcinomas, however, the mechanisms by which tumor cells ultimately die are considerably more complex. Although the machinery of apoptosis is engaged by toxic stimuli, other processes such as autophagy ("self-eating") and replicative cell death can lead to observations that do not simplistically correspond to any of the individual Type I-III formalized death categories. The 'hybrid' forms of cell death observed in carcinoma cells result in cellular materials being released into the extracellular space without packaging, which promotes inflammation, potentially leading to the accelerated re-growth of surviving tumor cells by macrophages. Drugs as single agents or in combinations can simultaneously initiate signaling via both apoptotic and autophagic pathways. Based on the tumor type and its oncogene drivers, as well as the drug(s) being used and the duration and intensity of the autophagosome signal, apoptosis and autophagy have the potential to act in concert to kill or alternatively that the actions of either pathway can act to suppress signaling by the other pathway. And, there also is evidence that autophagic flux, by causing lysosomal protease activation, with their subsequent release into the cytosol, can directly mediate killing. This review will discuss the interactive biology between apoptosis and autophagy in carcinoma cells. Finally, the molecular actions of the FDA-approved drugs neratinib and sorafenib, and how they enhance both apoptotic and toxic autophagic processes, alone or in combination with other agents, is discussed in a bench-to-bedside manner.

The development of multi-kinase inhibitors as pancreatic cancer therapeutics.

Pancreatic cancer is an almost incurable malignancy whose incidence has increased over the past 30 years. Instead of pursuing the development of modalities utilizing 'traditional' cytotoxic chemotherapeutic agents, we have explored the possibilities of developing novel multi-kinase inhibitor drug combinations to kill this tumor type. Several approaches using the multi-kinase inhibitors sorafenib, regorafenib, and neratinib have been safely translated from the bench to the bedside, with objective anti-tumor responses. This review will discuss our prior preclinical and clinical studies and discuss future clinical opportunities in this disease.

(Curcumin+sildenafil) enhances the efficacy of 5FU and anti-PD1 therapies in vivo.

We have extended our analyses of (curcumin+sildenafil) biology. The drug combination caused vascularization and degradation of mutant K-RAS that correlated with reduced phosphorylation of ERK1/2, AKT T308, mTORC1, mTORC2, ULK1 S757, STAT3, STAT5, and NFκB and increased phosphorylation of eIF2α, ATM, AMPKα, ULK1 S317; all concomitant with elevated ATG13 S318 phosphorylation and autophagosome formation. Prior studies with drug combinations utilizing sildenafil have delineated an ATM-AMPK-ULK1 S317 pathway and an AKT-mTOR-ULK1 S757 pathway as modules which control ATG S318 phosphorylation and autophagosome formation. The knockdown of PKG reduced cell killing as well as reducing drug-enhanced phosphorylation of ATM, AMPKα, and ATG13. In the absence of PKG, no significant increase in ULK1 S317 phosphorylation was observed. In a Beclin1-dependent fashion, the drug combination reduced the expression of multiple histone deacetylase (HDAC) proteins, including HDAC2 and HDAC3. Molecular knockdown of HDAC2, HDAC3, and especially (HDAC2+HDAC3) significantly reduced the expression of PD-L1 and elevated expression of Class I human major histocompatibility complex. In vivo, (curcumin+sildenafil) enhanced the efficacy of 5-flurouracil against CT26 colorectal tumors. Prior exposure of established CT26 tumors to (curcumin+sildenafil) significantly enhanced the efficacy of a subsequently administered anti-PD-1 antibody. Collectively our data argue that (curcumin+sildenafil) has the potential in several settings to be an efficacious neoadjuvant therapy for colon cancer.

GZ17-6.02 initiates DNA damage causing autophagosome-dependent HDAC degradation resulting in enhanced anti-PD1 checkpoint inhibitory antibody efficacy.

Our studies examined the molecular mechanisms by which the novel cancer therapeutic GZ17-6.02 (NCT03775525) killed GI tumor cells. TZ17-6.02 activated ATM which was responsible for increased phosphorylation of nuclear γH2AX and AMPKα T172. ATM-AMPK signaling was responsible for the subsequent inactivation of mTORC1 and mTORC2, dephosphorylation of ULK1 S757, and increased phosphorylation of ULK1 S317 and of ATG13 S318, which collectively caused enhanced autophagosome formation. GZ17-6.02 interacted with 5-fluorouracil in an additive to greater than additive fashion to kill all of the tested GI tumor cell types. This was associated with greater ATM activation and a greater mammalian target of rapamycin inactivation and autophagosome induction. As a result, autophagy-dependent degradation of multiple histone deacetylase (HDAC) proteins and chaperone proteins occurred. Loss of HDAC expression was causal in reduced expression of programed death ligand 1 (PD-L1), ornithine decarboxylase, and indole amine 2,3-dioxygenase (IDO1) and in the elevated expression of major histocompatibility complex Class IA (MHCA). Treatment with GZ17-6.02 also resulted in enhanced efficacy of a subsequently administered anti-PD1 checkpoint inhibitory antibody. Thus, the primary mode of GZ17-6.02 action is to induce a DNA damage response concomitant with ATM activation, that triggers a series of interconnected molecular events that result in tumor cell death and enhanced immunogenicity.

Neratinib degrades MST4 via autophagy that reduces membrane stiffness and is essential for the inactivation of PI3K, ERK1/2, and YAP/TAZ signaling.

The irreversible ERBB1/2/4 inhibitor neratinib causes plasma membrane-associated K-RAS to mislocalize into intracellular vesicles liminal to the plasma membrane; this effect is enhanced by HDAC inhibitors and is now a Phase I trial (NCT03919292). The combination of neratinib and HDAC inhibitors killed pancreatic cancer and lymphoma T cells. Neratinib plus HDAC inhibitor exposure was as efficacious as (paclitaxel+gemcitabine) at killing pancreatic cancer cells. Neratinib reduced the phosphorylation of PAK1, Merlin, LATS1/2, AKT, mTOR, p70 S6K, and ERK1/2 which required expression of Rubicon, Beclin1, and Merlin. Neratinib altered pancreatic tumor cell morphology which was associated with MST4 degradation reduced Ezrin phosphorylation and enhanced phosphorylation of MAP4K4 and LATS1/2. Knockdown of the MAP4K4 activator and sensor of membrane rigidity RAP2A reduced basal LATS1/2 and YAP phosphorylation but did not prevent neratinib from stimulating LATS1/2 or YAP phosphorylation. Beclin1 knockdown prevented MST4 degradation, Ezrin dephosphorylation and neratinib-induced alterations in tumor cell morphology. Our findings demonstrate that neratinib enhances LATS1/2 phosphorylation independently of RAP2A/MAP4K4 and that MST4 degradation and Ezrin dephosphorylation may represent a universal trigger for the biological actions of neratinib.

Neratinib augments the lethality of [regorafenib + sildenafil].

Regorafenib is approved for the treatment of colorectal cancer and hepatocellular carcinoma. In the trial NCT02466802, we have discovered that regorafenib can be safely combined with the phosphodiesterase 5 inhibitor sildenafil in advanced solid tumor patients. The present studies determined whether the approved ERBB1/2/4 and RAS downregulating drug neratinib, could enhance the lethality of [regorafenib + sildenafil]. Neratinib enhanced [regorafenib + sildenafil] lethality in a greater than additive fashion in colon cancer cells. The drug combination reduced the expression of mutant K-RAS and of multiple histone deacetylase (HDAC) proteins that required autophagosome formation. It caused green fluorescent protein or red fluorescent protein-tagged forms of K-RAS V12 to localize into large intracellular vesicles. Compared with [regorafenib + sildenafil], the three-drug combination caused greater and more prolonged activation of the ATM-AMPK-ULK-1 pathway and caused a greater suppression and prolonged inactivation of mammalian target of rapamycin, AKT, and p70 S6K. Approximately 70% of enhanced lethality caused by neratinib required ataxia-telangiectasia-mutated (ATM)-AMP-dependent protein kinase (AMPK) signaling whereas knockdown of Beclin1, ATG5, FADD, and CD95 completely prevented the elevated killing effect. Exposure of cells to [regorafenib + sildenafil] reduced the expression of the checkpoint immunotherapy biomarkers programmed death-ligand 1, ornithine decarboxylase, and indoleamine 2,3-dioxygenase-1 and increased the expression of major histocompatibility complex A (MHCA), which also required autophagosome formation. Knockdown of specific HDAC proteins recapitulated the effects observed using chemical agents. In vivo, using mouse cancer models, neratinib significantly enhanced the antitumor efficacy of [regorafenib + sildenafil]. Our data support performing a new three drug Phase I trial combining regorafenib, sildenafil, and neratinib.

AR-12 Inhibits Multiple Chaperones Concomitant With Stimulating Autophagosome Formation Collectively Preventing Virus Replication.

We have recently demonstrated that AR-12 (OSU-03012) reduces the function and ATPase activities of multiple HSP90 and HSP70 family chaperones. Combined knock down of chaperones or AR-12 treatment acted to reduce the expression of virus receptors and essential glucosidase proteins. Combined knock down of chaperones or AR-12 treatment inactivated mTOR and elevated ATG13 S318 phosphorylation concomitant with inducing an endoplasmic reticulum stress response that in an eIF2α-dependent fashion increased Beclin1 and LC3 expression and autophagosome formation. Over-expression of chaperones prevented the reduction in receptor/glucosidase expression, mTOR inactivation, the ER stress response, and autophagosome formation. AR-12 reduced the reproduction of viruses including Mumps, Influenza, Measles, Junín, Rubella, HIV (wild type and protease resistant), and Ebola, an effect replicated by knock down of multiple chaperone proteins. AR-12-stimulated the co-localization of Influenza, EBV and HIV virus proteins with LC3 in autophagosomes and reduced viral protein association with the chaperones HSP90, HSP70, and GRP78. Knock down of Beclin1 suppressed drug-induced autophagosome formation and reduced the anti-viral protection afforded by AR-12. In an animal model of hemorrhagic fever virus, a transient exposure of animals to low doses of AR-12 doubled animal survival from ∼30% to ∼60% and suppressed liver damage as measured by ATL, GGT and LDH release. Thus through inhibition of chaperone protein functions; reducing the production, stability and processing of viral proteins; and stimulating autophagosome formation/viral protein degradation, AR-12 acts as a broad-specificity anti-viral drug in vitro and in vivo. We argue future patient studies with AR-12 are warranted. J. Cell. Physiol. 231: 2286-2302, 2016. © 2016 Wiley Periodicals, Inc.

Nexavar/Stivarga and viagra interact to kill tumor cells.

We determined whether the multi-kinase inhibitor sorafenib or its derivative regorafenib interacted with phosphodiesterase 5 (PDE5) inhibitors such as Viagra (sildenafil) to kill tumor cells. PDE5 and PDGFRα/ß were over-expressed in liver tumors compared to normal liver tissue. In multiple cell types in vitro sorafenib/regorafenib and PDE5 inhibitors interacted in a greater than additive fashion to cause tumor cell death, regardless of whether cells were grown in 10 or 100% human serum. Knock down of PDE5 or of PDGFRα/ß recapitulated the effects of the individual drugs. The drug combination increased ROS/RNS levels that were causal in cell killing. Inhibition of CD95/FADD/caspase 8 signaling suppressed drug combination toxicity. Knock down of ULK-1, Beclin1, or ATG5 suppressed drug combination lethality. The drug combination inactivated ERK, AKT, p70 S6K, and mTOR and activated JNK. The drug combination also reduced mTOR protein expression. Activation of ERK or AKT was modestly protective whereas re-expression of an activated mTOR protein or inhibition of JNK signaling almost abolished drug combination toxicity. Sildenafil and sorafenib/regorafenib interacted in vivo to suppress xenograft tumor growth using liver and colon cancer cells. From multiplex assays on tumor tissue and plasma, we discovered that increased FGF levels and ERBB1 and AKT phosphorylation were biomarkers that were directly associated with lower levels of cell killing by 'rafenib + sildenafil. Our data are now being translated into the clinic for further determination as to whether this drug combination is a useful anti-tumor therapy for solid tumor patients.

OSU-03012 and Viagra Treatment Inhibits the Activity of Multiple Chaperone Proteins and Disrupts the Blood-Brain Barrier: Implications for Anti-Cancer Therapies.

We examined the interaction between OSU-03012 (also called AR-12) with phosphodiesterase 5 (PDE5) inhibitors to determine the role of the chaperone glucose-regulated protein (GRP78)/BiP/HSPA5 in the cellular response. Sildenafil (Viagra) interacted in a greater than additive fashion with OSU-03012 to kill stem-like GBM cells. Treatment of cells with OSU-03012/sildenafil: abolished the expression of multiple oncogenic growth factor receptors and plasma membrane drug efflux pumps and caused a rapid degradation of GRP78 and other HSP70 and HSP90 family chaperone proteins. Decreased expression of plasma membrane receptors and drug efflux pumps was dependent upon enhanced PERK-eIF2α-ATF4-CHOP signaling and was blocked by GRP78 over-expression. In vivo OSU-03012/sildenafil was more efficacious than treatment with celecoxib and sildenafil at killing tumor cells without damaging normal tissues and in parallel reduced expression of ABCB1 and ABCG2 in the normal brain. The combination of OSU-03012/sildenafil synergized with low concentrations of sorafenib to kill tumor cells, and with lapatinib to kill ERBB1 over-expressing tumor cells. In multiplex assays on plasma and human tumor tissue from an OSU-03012/sildenafil treated mouse, we noted a profound reduction in uPA signaling and identified FGF and JAK1/2 as response biomarkers for potentially suppressing the killing response. Inhibition of FGFR signaling and to a lesser extent JAK1/2 signaling profoundly enhanced OSU-03012/sildenafil lethality.

PDE5 inhibitors enhance celecoxib killing in multiple tumor types.

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with a clinically relevant NSAID, celecoxib, to kill tumor cells. Celecoxib and PDE5 inhibitors interacted in a greater than additive fashion to kill multiple tumor cell types. Celecoxib and sildenafil killed ex vivo primary human glioma cells as well as their associated activated microglia. Knock down of PDE5 recapitulated the effects of PDE5 inhibitor treatment; the nitric oxide synthase inhibitor L-NAME suppressed drug combination toxicity. The effects of celecoxib were COX2 independent. Over-expression of c-FLIP-s or knock down of CD95/FADD significantly reduced killing by the drug combination. CD95 activation was dependent on nitric oxide and ceramide signaling. CD95 signaling activated the JNK pathway and inhibition of JNK suppressed cell killing. The drug combination inactivated mTOR and increased the levels of autophagy and knock down of Beclin1 or ATG5 strongly suppressed killing by the drug combination. The drug combination caused an ER stress response; knock down of IRE1α/XBP1 enhanced killing whereas knock down of eIF2α/ATF4/CHOP suppressed killing. Sildenafil and celecoxib treatment suppressed the growth of mammary tumors in vivo. Collectively our data demonstrate that clinically achievable concentrations of celecoxib and sildenafil have the potential to be a new therapeutic approach for cancer.

GRP78/Dna K Is a Target for Nexavar/Stivarga/Votrient in the Treatment of Human Malignancies, Viral Infections and Bacterial Diseases.

Prior tumor cell studies have shown that the drugs sorafenib (Nexavar) and regorafenib (Stivarga) reduce expression of the chaperone GRP78. Sorafenib/regorafenib and the multi-kinase inhibitor pazopanib (Votrient) interacted with sildenafil (Viagra) to further rapidly reduce GRP78 levels in eukaryotes and as single agents to reduce Dna K levels in prokaryotes. Similar data were obtained in tumor cells in vitro and in drug-treated mice for: HSP70, mitochondrial HSP70, HSP60, HSP56, HSP40, HSP10, and cyclophilin A. Prolonged 'rafenib/sildenafil treatment killed tumor cells and also rapidly decreased the expression of: the drug efflux pumps ABCB1 and ABCG2; and NPC1 and NTCP, receptors for Ebola/Hepatitis A and B viruses, respectively. Pre-treatment with the 'Rafenib/sildenafil combination reduced expression of the Coxsackie and Adenovirus receptor in parallel with it also reducing the ability of a serotype 5 Adenovirus or Coxsackie virus B4 to infect and to reproduce. Sorafenib/pazopanib and sildenafil was much more potent than sorafenib/pazopanib as single agents at preventing Adenovirus, Mumps, Chikungunya, Dengue, Rabies, West Nile, Yellow Fever, and Enterovirus 71 infection and reproduction. 'Rafenib drugs/pazopanib as single agents killed laboratory generated antibiotic resistant E. coli which was associated with reduced Dna K and Rec A expression. Marginally toxic doses of 'Rafenib drugs/pazopanib restored antibiotic sensitivity in pan-antibiotic resistant bacteria including multiple strains of blakpc Klebsiella pneumoniae. Thus, Dna K is an antibiotic target for sorafenib, and inhibition of GRP78/Dna K has therapeutic utility for cancer and for bacterial and viral infections.

GRP78/BiP/HSPA5/Dna K is a universal therapeutic target for human disease.

The chaperone GRP78/Dna K is conserved throughout evolution down to prokaryotes. The GRP78 inhibitor OSU-03012 (AR-12) interacted with sildenafil (Viagra) or tadalafil (Cialis) to rapidly reduce GRP78 levels in eukaryotes and as a single agent reduce Dna K levels in prokaryotes. Similar data with the drug combination were obtained for: HSP70, HSP90, GRP94, GRP58, HSP27, HSP40 and HSP60. OSU-03012/sildenafil treatment killed brain cancer stem cells and decreased the expression of: NPC1 and TIM1; LAMP1; and NTCP1, receptors for Ebola/Marburg/Hepatitis A, Lassa fever, and Hepatitis B viruses, respectively. Pre-treatment with OSU-03012/sildenafil reduced expression of the coxsakie and adenovirus receptor in parallel with it also reducing the ability of a serotype 5 adenovirus or coxsakie virus B4 to infect and to reproduce. Similar data were obtained using Chikungunya, Mumps, Measles, Rubella, RSV, CMV, and Influenza viruses. OSU-03012 as a single agent at clinically relevant concentrations killed laboratory generated antibiotic resistant E. coli and clinical isolate multi-drug resistant N. gonorrhoeae and MRSE which was in bacteria associated with reduced Dna K and Rec A expression. The PDE5 inhibitors sildenafil or tadalafil enhanced OSU-03012 killing in N. gonorrhoeae and MRSE and low marginally toxic doses of OSU-03012 could restore bacterial sensitivity in N. gonorrhoeae to multiple antibiotics. Thus, Dna K and bacterial phosphodiesterases are novel antibiotic targets, and inhibition of GRP78 is of therapeutic utility for cancer and also for bacterial and viral infections.

Phosphodiesterase 5 inhibitors enhance chemotherapy killing in gastrointestinal/genitourinary cancer cells.

The present studies determined whether clinically relevant phosphodiesterase 5 (PDE5) inhibitors interacted with clinically relevant chemotherapies to kill gastrointestinal/genitourinary cancer cells. In bladder cancer cells, regardless of H-RAS mutational status, at clinically achievable doses, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/mitomycin C/gemcitabine/cisplatin/paclitaxel to cause cell death. In pancreatic tumor cells expressing mutant active K-RAS, PDE5 inhibitors interacted in a greater than additive fashion with doxorubicin/gemcitabine/paclitaxel to cause cell death. The most potent PDE5 inhibitor was sildenafil. Knock down of PDE5 expression recapitulated the combination effects of PDE5 inhibitor drugs with chemotherapy drugs. Expression of cellular FLICE-like inhibitory protein-short did not significantly inhibit chemotherapy lethality but did significantly reduce enhanced killing in combination with sildenafil. Overexpression of B-cell lymphoma-extra large suppressed individual and combination drug toxicities. Knock down of CD95 or Fas-associated death domain protein suppressed drug combination toxicity. Combination toxicity was also abolished by necrostatin or receptor interacting protein 1 knock down. Treatment with PDE5 inhibitors and chemotherapy drugs promoted autophagy, which was maximal at ∼24 hour posttreatment, and 3-methyl adenine or knock down of Beclin1 suppressed drug combination lethality by ∼50%. PDE5 inhibitors enhanced and prolonged the induction of DNA damage as judged by Comet assays and γhistone 2AX (γH2AX) and checkpoint kinase 2 (CHK2) phosphorylation. Knock down of ataxia telangiectasia mutated suppressed γH2AX and CHK2 phosphorylation and enhanced drug combination lethality. Collectively our data demonstrate that the combination of PDE5 inhibitors with standard of care chemotherapy agents for gastrointestinal/genitourinary cancers represents a novel modality.

GZ17-6.02 interacts with proteasome inhibitors to kill multiple myeloma cells.

GZ17-6.02, a synthetically manufactured compound containing isovanillin, harmine and curcumin, has undergone phase I evaluation in patients with solid tumors (NCT03775525) with a recommended phase 2 dose (RP2D) of 375 mg PO BID. GZ17-6.02 was more efficacious as a single agent at killing multiple myeloma cells than had previously been observed in solid tumor cell types. GZ17-6.02 interacted with proteasome inhibitors in a greater than additive fashion to kill myeloma cells and alone it killed inhibitor-resistant cells to a similar extent. The drug combination of GZ17-6.02 and bortezomib activated ATM, the AMPK and PERK and inactivated ULK1, mTORC1, eIF2α, NFκB and the Hippo pathway. The combination increased ATG13 S318 phosphorylation and the expression of Beclin1, ATG5, BAK and BIM, and reduced the levels of BCL-XL and MCL1. GZ17-6.02 interacted with bortezomib to enhance autophagosome formation and autophagic flux, and knock down of ATM, AMPKα, ULK1, Beclin1 or ATG5 significantly reduced both autophagy and tumor cell killing. Knock down of BAK and BIM significantly reduced tumor cell killing. The expression of HDACs1/2/3 was significantly reduced beyond that previously observed in solid tumor cells and required autophagy. This was associated with increased acetylation and methylation of histone H3. Combined knock down of HDACs1/2/3 caused activation of ATM and the AMPK and caused inactivation of ULK1, mTORC1, NFκB and the Hippo pathway. HDAC knock down also enhanced ATG13 phosphorylation, increased BAK levels and reduced those of BCL-XL. Collectively, our present studies support performing additional in vivo studies with multiple myeloma cells.

GZ17-6.02 interacts with bexarotene to kill mycosis fungoides cells.

GZ17-6.02, composed of curcumin, harmine and isovanillin, has undergone phase I evaluation in patients with solid tumors (NCT03775525) with an RP2D of 375 mg PO BID. The biology of GZ17-6.02 in malignant T cells and in particular those derived from mycosis fungoides (MF) patients, has not been studied. GZ17-6.02 alone and in combination with standard-of-care agents was effective in killing MF cells. All three components are necessary for optimal killing of MF cells. GZ17-6.02 activated ATM, the AMPK, NFκB and PERK and inactivated ERK1/2, AKT, ULK1, mTORC1, eIF2α, and reduced the expression of BCL-XL and MCL1. GZ17-6.02 increased ATG13 S318 phosphorylation and the expression of Beclin1, ATG5, BAK and BIM. GZ17-6.02 in a dose-dependent fashion enhanced autophagosome formation and autophagic flux, and tumor cell killing. Signaling by ATM and AMPK were both required for efficient killing but not for the dose-response effect whereas ER stress (eIF2α) and macroautophagy (Beclin1, ATG5) were required for both efficient killing and the dose-response. Knock down of the death receptor CD95 reduced killing by ~20% and interacted with autophagy inhibition to further reduce killing, collectively, by ~70%. Inhibition of autophagy and knock down of death-mediators downstream of the mitochondrion, AIF and caspase 3, almost abolished tumor cell killing. Hence in MF cells, GZ17-6.02 is a multi-factorial killer, utilizing ER stress, macroautophagy, death receptor signaling and directly causing mitochondrial dysfunction.

GZ17-6.02 kills PDX isolates of uveal melanoma.

GZ17-6.02 has undergone phase I evaluation in patients with solid tumors (NCT03775525). The RP2D is 375 mg PO BID, with an uveal melanoma patient exhibiting a 15% reduction in tumor mass for 5 months at this dose. Studies in this manuscript have defined the biology of GZ17-6.02 in PDX isolates of uveal melanoma cells. GZ17-6.02 killed uveal melanoma cells through multiple convergent signals including enhanced ATM-AMPK-mTORC1 activity, inactivation of YAP/TAZ and inactivation of eIF2α. GZ17-6.02 significantly enhanced the expression of BAP1, predictive to reduce metastasis, and reduced the levels of ERBB family RTKs, predicted to reduce growth. GZ17-6.02 interacted with doxorubicin or ERBB family inhibitors to significantly enhance tumor cell killing which was associated with greater levels of autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5 or eIF2α were more protective than knock down of ATM, AMPKα, CD95 or FADD, however, over-expression of FLIP-s provided greater protection compared to knock down of CD95 or FADD. Expression of activated forms of mTOR and STAT3 significantly reduced tumor cell killing. GZ17-6.02 reduced the expression of PD-L1 in uveal melanoma cells to a similar extent as observed in cutaneous melanoma cells whereas it was less effective at enhancing the levels of MHCA. The components of GZ17-6.02 were detected in tumors using a syngeneic tumor model. Our data support future testing GZ17-6.02 in uveal melanoma as a single agent, in combination with ERBB family inhibitors, in combination with cytotoxic drugs, or with an anti-PD1 immunotherapy.