Protein-protein interactions featuring intricate binding epitopes remain challenging targets for synthetic inhibitors. Interactions of NEMO, a scaffolding protein central to NF-κB signaling, ...exemplify this challenge. Various regulators are known to interact with different coiled coil regions of NEMO, but the topological complexity of this protein has limited inhibitor design. We undertook a comprehensive effort to block the interaction between vFLIP, a Kaposi's sarcoma herpesviral oncoprotein, and NEMO using small molecule screening and rational design. Our efforts reveal that a tertiary protein structure mimic of NEMO is necessary for potent inhibition. The rationally designed mimic engages vFLIP directly causing complex disruption, protein degradation and suppression of NF-κB signaling in primary effusion lymphoma (PEL). NEMO mimic treatment induces cell death and delays tumor growth in a PEL xenograft model. Our studies with this inhibitor reveal the critical nexus of signaling complex stability in the regulation of NF-κB by a viral oncoprotein.
Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets ...the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity
and
, including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition
and
Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR.
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During lytic herpes simplex virus (HSV) infections, the virion host shutoff (Vhs) (UL41) endoribonuclease degrades many cellular and viral mRNAs. In uninfected cells, spliced mRNAs emerge into the ...cytoplasm bound by exon junction complexes (EJCs) and are translated several times more efficiently than unspliced mRNAs that have the same sequence but lack EJCs. Notably, most cellular mRNAs are spliced, whereas most HSV mRNAs are not. To examine the effect of splicing on gene expression during HSV infection, cells were transfected with plasmids harboring an unspliced renilla luciferase (RLuc) reporter mRNA or RLuc constructs with introns near the 5' or 3' end of the gene. After splicing of intron-containing transcripts, all three RLuc mRNAs had the same primary sequence. Upon infection in the presence of actinomycin D, spliced mRNAs were much less sensitive to degradation by copies of Vhs from infecting virions than were unspliced mRNAs. During productive infections (in the absence of drugs), RLuc was expressed at substantially higher levels from spliced than from unspliced mRNAs. Interestingly, the stimulatory effect of splicing on RLuc expression was significantly greater in infected than in uninfected cells. The translational stimulatory effect of an intron during HSV-1 infections could be replicated by artificially tethering various EJC components to an unspliced RLuc transcript. Thus, the splicing history of an mRNA, and the consequent presence or absence of EJCs, affects its level of translation and sensitivity to Vhs cleavage during lytic HSV infections.
Most mammalian mRNAs are spliced. In contrast, of the more than 80 mRNAs harbored by herpes simplex virus 1 (HSV-1), only 5 are spliced. In addition, synthesis of the immediate early protein ICP27 causes partial inhibition of pre-mRNA splicing, with the resultant accumulation of both spliced and unspliced versions of some mRNAs in the cytoplasm. A common perception is that HSV-1 infection necessarily inhibits the expression of spliced mRNAs. In contrast, this study demonstrates two instances in which pre-mRNA splicing actually enhances the synthesis of proteins from mRNAs during HSV-1 infections. Specifically, splicing stabilized an mRNA against degradation by copies of the Vhs endoribonuclease from infecting virions and greatly enhanced the amount of protein synthesized from spliced mRNAs at late times after infection. The data suggest that splicing, and the resultant presence of exon junction complexes on an mRNA, may play an important role in gene expression during HSV-1 infections.
e15097 Background: Denosumab is a RANKL inhibitor used in patients with bone metastases from solid tumors. FKS518 is a proposed biosimilar to denosumab originator product. Biosimilars offer similar ...efficacy, quality, safety, and favorable economic impact. Once the analytical and functional similarity of a biosimilar has been shown, a comparative human pharmacokinetics (PK) study is conducted to demonstrate that there are no clinically meaningful differences between the biosimilar and the reference product. The present study assessed the PK equivalence of FKS518 to the originator product and provided data on the similarity of the pharmacodynamic, safety and immunogenicity profiles. Methods: A randomized, controlled, double-blind, parallel-group study in healthy volunteers was performed to compare denosumab biosimilar candidate FKS518 versus the originator product. Subjects received a single subcutaneous denosumab 60 mg injection and were followed up for 40 weeks. Primary endpoints PK parameters (Cmax, AUC0-last and AUC0-inf) were natural log transformed and analyzed using analysis of covariance methods; results were then transformed back to the original scale. Safety data were collected throughout the study and analyzed descriptively. Results: 213 male subjects 28-55 years of age, with a baseline BMI between 18.2 and 31.7 kg/m2, were randomized to the originator denosumab (n = 106) or FK518 (n = 107). Results of the primary PK analysis demonstrated bioequivalence between FKS518 and the originator denosumab product since the 90% confidence intervals (CIs) for the geometric least squares mean (LSM) ratios for the 3 primary PK parameters were completely included within the predefined 80.00% to 125.00% equivalence margin (point estimate 90%CI for the ratio): Cmax: 104.79% 97.04%,113.15%, AUC0-last: 112.29% 104.17%,121.04% and AUC0-inf : 112.65% 104.27%,121.70%. Secondary PK parameters were similar as well between FKS518 and the originator: tmax (median: 217.45 hrs. vs. 216.03 hrs. respectively), t1/2 (median: 235 hrs. vs 337 hrs. respectively). The nature, frequency, severity, or resolution of adverse events were comparable across treatments. For immunogenicity, there was no difference between the 2 treatment groups in ADA and Nab incidence at each timepoint. Conclusions: PK equivalence of FKS518 and denosumab originator product was demonstrated with similar PD, safety and immunogenicity profile for the two products. This study adds to the totality of evidence supporting FKS518 as a proposed biosimilar to denosumab.
3155 Background: Biosimilars can increase patient access to treatment. FKS518 is a candidate biosimilar of denosumab, a RANKL inhibitor used in patients with bone metastases from certain solid tumors ...and in multiple myeloma. To establish biosimilarity, comparative studies are conducted in the most sensitive patient population to detect if there are clinically meaningful differences. LUMIADE-3 was designed to assess the safety and efficacy of FKS518 compared to the originator in postmenopausal women with osteoporosis (PMO). Methods: A randomized, double-blind, multicenter, 2-arm study recruited female patients aged 55 to 85 years with confirmed postmenopausal status and lumbar spine bone mineral density (LS-BMD) T-score ≤ -2.5 and ≥ -4.0, as measured by central dual-energy X-ray absorptiometry (DXA) assessment. Subjects with prior osteoporosis treatment that could add risk for cumulative effect or affect the interpretation of results were excluded. Patients were randomized and received three 60 mg administrations. At week 52, patients receiving denosumab originator were re-randomized to continue their treatment or switch to FKS518 for the third dose. Those receiving FKS518 continued receiving FKS518. The primary efficacy endpoint was the percent change from baseline in LS-BMD DXA at week 52. An analysis of the covariance model was used to compare the two treatments and two separate one-sided tests at alpha=0.05 were performed to assess equivalence. Secondary efficacy endpoints included the percent change from baseline in BMD at the femoral neck and total hip. Results: A total of 553 patients were randomized to FKS518 (n= 276) or the originator (n=277). Clinically relevant increases in LS-BMD were evident at week 52 in both the FKS518 and originator product groups. The results demonstrated therapeutic equivalence: the lower bound of the 90% CI for non-inferiority (-0.05) was above -1.45, and the upper bound of the 90% CI for non-superiority (1.20) was below 1.45. All secondary objectives were met. At week 52, a similar percent change from baseline in BMD at the femoral neck and total hip was observed between the two groups. 374 patients (67.6%) experienced treatment-emergent adverse events: 185 (66.8%) in the FKS518 group and 189 (68.5%) in the originator product group. Safety evaluation did not point to notable differences between FKS518 and the originator product groups. Conclusions: We demonstrated the therapeutic equivalence of FKS518 and originator product. The safety data showed similar safety profiles for the FKS518 and the originator product groups. Results from this study add to the totality of evidence supporting the similarity of FKS518 as a proposed biosimilar to denosumab originator product, a RANKL inhibitor used in patients with bone metastases from solid tumors. Clinical trial information: NCT04934072 .
Although nucleoside analogues have been used effectively in the clinic for the treatment of a wide range of hematological malignancies, lack of response to currently available nucleoside analogues ...and drug resistance limit their utility. A rare but highly aggressive cancer is primary effusion lymphoma (PEL). Through high throughput screening, we have discovered a novel nucleoside analog, called 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, with little activity in other lymphomas tested. PEL is a rare B-cell non-Hodgkin's lymphoma characterized by lymphomatous effusions in body cavities. It is associated with Kaposi's sarcoma herpesvirus (KSHV/HHV-8) infection and occurs mainly in immunocompromised patients. PEL is known to frequently be resistant to conventional chemotherapy (CHOP and EPOCH) resulting in poor prognosis and a rather incurable disease. Our studies demonstrated that 6-ETI is a pro-drug activated by adenosine kinase (ADK), an enzyme that is overexpressed in PEL cell lines and primary PEL specimens, as well as other plasma cell malignancies, including plasmablastic lymphoma (PBL) and multiple myeloma (MM). The latter is also responsive to 6-ETI in vitro and in mouse models. 6-ETI induces S phase arrest and inhibits DNA synthesis. RNA sequencing of in vitro generated PEL resistant clones and CRISPR knock out of ADK (ADK KO), respectively, indicated that mutations or loss of expression of ADK renders cells resistant to treatment. This data demonstrates that ADK expression can be used as a predictive biomarker of response to 6-ETI, which can help identify which patients are more likely to respond to this treatment.
We investigated which pathways are differentially regulated in sensitive and resistant cells to better delineate the mechanism of action of 6-ETI and to design effective combinatorial regimens and prevent resistance. We found that drug sensitivity was associated with AMPK activation and inhibition of PI3K/mTOR/p70S6K signaling. Little is known about the function of ADK in plasma cell neoplasms. Knock-out of this protein in PEL, or use of ADK chemical inhibitors, do not affect their viability. Thus, we used ADK KO cell lines to examine the role of ADK in these tumors and to determine if cells undergo adaptations that may contribute to 6-ETI resistance and represent potential vulnerabilities to combat it. We performed metabolic and transcriptomic profiling of wild type (WT) (6-ETI sensitive) and ADK KO (6-ETI resistant) cells to achieve a comprehensive assessment of all the metabolic perturbations and gene expression changes induced by knocking out ADK. We also treated these cells with 6-ETI to examine the effects in sensitive and resistant cells. This integrated analysis revealed that 6-ETI depletes sensitive PEL cells of their nucleotide pools accompanied by the downregulation of several genes in purine and pyrimidine biosynthesis pathways. We found that adenine supplementation rescues sensitive PEL cells from 6-ETI induced cytotoxicity, reverses p70S6K inhibition and restores DNA synthesis suggesting that purine metabolism is a critical mediator of 6-ETI induced cytotoxicity. Using seahorse bioenergetic assay, we show that ADK KO resistant cells have impaired mitochondrial respiration indicating that ADK plays a critical role in mitochondrial bioenergetics. Metabolic profiling of these ADK KO resistant cells showed that these cells have elevated levels of de novo pyrimidine metabolic intermediates. Metabolic flux through de novo pyrimidine is controlled by the rate limiting enzyme CAD. The activity of CAD is regulated by ribosomal protein S6 Kinase 1(S6K1) by phosphorylation at its (Ser1859) site. Using western blotting, we observed a striking increase of phosphorylation of CAD at its S6K1 site (Ser1859) in ADK KO cells compared to WT cells.
This is the first to date study that characterizes the role of ADK in lymphomas. Our data indicates that ADK KO cells have undergone metabolic reprogramming to upregulate de novo pyrimidine biosynthesis and p70S6K signaling. Moreover, we found that 6-ETI synergizes with the pan PI3K inhibitor BKM120 highlighting nucleotide metabolism and PI3K/mTOR signaling as key therapeutic vulnerabilities targeted by this novel nucleoside analog.
No relevant conflicts of interest to declare.
Introduction: Previous gene expression profiling studies of classical Hodgkin lymphoma (cHL) have been confined to cell lines and microdissected HRS cells from tissue biopsies given the difficulty of ...isolating sparse Hodgkin and Reed-Sternberg (HRS) cells from reactive background tissue. We previously used flow sorting to separate HRS cells from fresh or viably frozen cHL biopsies, and performed the first full exome sequencing of HRS cells. Here we report use of the same cell separation approach to examine the HRS cell transcriptome using RNA sequencing.
Methods: We used flow cytometric cell sorting and low-input RNA sequencing to generate full transcriptome data from viable primary HRS cells, along with intratumor B cells. Nine primary cases of cHL and four cell lines were assessed for RNA expression, expressed mutations, cell type of origin, signaling pathways, gene fusions and pathogen identification. We used immunohistochemistry to evaluate expression of PDIA6 and CD48 in the 9 cases sequenced and a tissue microarray containing 16 additional cases of cHL. Flow cytometry for CD48 was performed in two cell lines and 5 primary cases.
Results: Clustering show that primary HRS cells have a transcriptional profile that is unique, and different from that of intratumoral B cells, as well as cHL cell lines. Comparison of HRS cells with normal cellular subsets indicated plasma cell differentiation, suggesting that the cell of origin is a B cell on its way to becoming a plasma cell. Clustering with B cells showed much lower similarity. Consistent with plasma cell differentiation, we uncovered an unfolded protein response UPR) signature, shared with plasma cell neoplasms and, to a lesser extent, activated B cell (ABC) diffuse large B cell lymphoma, but not other B cell lymphoma types, including primary mediastinal B cell lymphoma (PMBCL). Among other UPR response genes, PDIA6 showed strong downregulation at the RNA level (2.4 logFC, p=9.4E-17). This finding was validated by immunohistochemistry for PDIA6, which showed strong positivity in the HRS cells of all 25 cases examined, confirming that this is a common feature of cHL, including nodular sclerosis and mixed cellularity subtypes.
Top upregulated genes included those involved in oncogenesis (HGF/MET, NFkB/apoptosis inhibition), stem cell differentiation (homeobox genes MEIS1 and PBX1), and mitotic checkpoints, mitotic spindle formation and DNA repair, possibly explaining the unique nuclear morphology of HRS cells. Downregulation of MHC-1 and MHC-2 driven antigen processing and presentation was confirmed, and so was overexpression of PDL1 (CD274). Importantly, we detected loss of SLAM family receptors, which serve as activation signals for NK cells providing an additional mechanism for tumor immune evasion. One of these is CD48 (-2.63 logFC, p=1.56E-05), which was confirmed to be strongly downregulated. This finding was confirmed by immunohistochemistry (25 cases) and flow cytometry (2 cell lines and 5 primary cases) on the expanded sample set. Given that only some cHL cases are associated with EBV infection, it has been speculated that other viruses are involved in negative cases. However, our analysis did not reveal additional viruses in the HRS cells.
Conclusions: Our data indicate that cHL more closely resembles plasma cells than B cells, and plasma cell malignancies than other lymphomas. The salient feature of plasmacytic differentiation is a UPR, which is seen in HRS cells and multiple myeloma. In contrast, UPR is not a feature of primary mediastinal B cell lymphoma, which is thought to be the DLBCL most similar to cHL clinically, immunophenotypically and in terms of gene expression patterns. We also provide an integrated view of potential immune evasion mechanisms by HRS cells that potentially explain lack of anti-tumor T or innate response. These include lack of antigen presentation due to B2M mutations, overexpression of PDL1 and PDL2, immunosuppressive cytokine secretion and, for the first time, a demonstration of lack of NK activating receptors of the SLAM family. Lack of SLAM family receptors may explain lack of NK cells clearance of HRS cells in the face of MHC-I downregulation. It has long been suspected that cHL is a tumor where there likely exists a previously undiscovered virus in addition to EBV, but RNA sequencing failed to reveal additional infectious transcripts in the HRS cells.
Roshal:Celgene: Other: Provision of Services; Auron Therapeutics: Equity Ownership, Other: Provision of services; Physicians' Education Resource: Other: Provision of services. Brody:Kite Pharma: Research Funding; Celldex Therapeutics: Research Funding; Genentech: Research Funding; Acerta Pharma: Research Funding; Oncovir, Inc.: Research Funding; BMS: Research Funding; Merck: Research Funding. Dave:Data Driven Bioscience: Equity Ownership.
Abstract
A number of nucleoside analogues are used successfully for the treatment of several cancers, and in particular leukemias and lymphomas, but these have distinct efficacies for different tumor ...types, and many malignancies do not respond to currently available nucleoside analogues or other forms of chemotherapy. A high throughput screen conducted in our lab to search for inhibitors of primary effusion lymphoma (PEL) identified the nucleoside analog 6-ethylthioinosine (6-ETI) as a potent and selective inhibitor of PEL, a largely incurable malignancy of B cell origin with plasmacytic differentiation. 6-ETI induced necrosis and ATP-depletion accompanied by S-phase arrest, DNA damage and inhibition of DNA synthesis. To understand 6-ETI mechanism of selectivity, RNA-seq analysis of in vitro generated drug-resistant PEL clones revealed inactivating mutations and loss of expression of adenosine kinase (ADK) as the mechanism of resistance. In vitro assays showed that 6-ETI is a pro-drug that gets phosphorylated and activated by adenosine kinase (ADK) into its active form. We found high ADK expression in PEL cell lines and primary specimens of PEL, multiple myeloma (MM) and plasmablastic lymphoma (PBL) patient samples. 6-ETI was effective at killing multiple myeloma cell lines, primary MM specimens, and had a remarkable anti-tumor response in a disseminated multiple myeloma and PEL xenograft mouse models. Thus, ADK expression can serve as a predictive biomarker to help identify patients that are most likely to respond to 6-ETI treatment. To further assess the spectrum of activity and sensitivity of 6-ETI, we examined ADK expression in other cancer subtypes and found that colorectal and pancreatic adenocarcinomas also overexpress ADK and are highly sensitive to killing by 6-ETI at the low nanomolar concentration. We also found high ADK expression in primary colon and pancreatic adenocarcinoma patient specimens. We compared 6-ETI to other FDA-approved purine analogs and failed to find other compounds with similar potency or selectivity profile. Herein, we report the identification of a novel purine analog, 6-ethylthioinosine, as an effective therapeutic with exquisite sensitivity to plasma cell malignancies and other ADK-expressing cancers. We have successfully used RNASeq-based “resistome” analysis to identify its mechanism of specificity and discovered a new biomarker that can potentially impact patient care and the treatment of some of the most aggressive tumors.
Citation Format: Jouliana Sadek, Utthara Nayar, Jonathan Reichel, Jennifer Totonchy, Shizuko Sei, Robert Shoemaker, David Warren, Olivier Elemento, Kenneth Kaye, Ethel Cesarman. A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas 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 5110. doi:10.1158/1538-7445.AM2017-5110
Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This ...compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase-inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI-sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.
Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin with plasmacytic differentiation. Here, we report the identification of a highly effective inhibitor of PEL. This ...compound, 6-ethylthioinosine (6-ETI), is a nucleoside analog with toxicity to PEL in vitro and in vivo, but not to other lymphoma cell lines tested. We developed and performed resistome analysis, an unbiased approach based on RNA sequencing of resistant subclones, to discover the molecular mechanisms of sensitivity. We found different adenosine kinase -inactivating (ADK-inactivating) alterations in all resistant clones and determined that ADK is required to phosphorylate and activate 6-ETI. Further, we observed that 6-ETI induces ATP depletion and cell death accompanied by S phase arrest and DNA damage only in ADK-expressing cells. Immunohistochemistry for ADK served as a biomarker approach to identify 6-ETI -sensitive tumors, which we documented for other lymphoid malignancies with plasmacytic features. Notably, multiple myeloma (MM) expresses high levels of ADK, and 6-ETI was toxic to MM cell lines and primary specimens and had a robust antitumor effect in a disseminated MM mouse model. Several nucleoside analogs are effective in treating leukemias and T cell lymphomas, and 6-ETI may fill this niche for the treatment of PEL, plasmablastic lymphoma, MM, and other ADK-expressing cancers.
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