Utthara Nayar

Targeting the Hsp90-associated viral oncoproteome in gammaherpesvirus-associated malignancies.

PU-H71 is a purine-scaffold Hsp90 inhibitor that, in contrast to other Hsp90 inhibitors, displays unique selectivity for binding the fraction of Hsp90 that is preferentially associated with oncogenic client proteins and enriched in tumor cells (teHsp90). This property allows PU-H71 to potently suppress teHsp90 without inducing toxicity in normal cells. We found that lymphoma cells infected by Epstein-Barr virus or Kaposi sarcoma-associated herpes virus (KSHV) are exquisitely sensitive to this compound. Using PU-H71 affinity capture and proteomics, an unbiased approach to reveal oncogenic networks, we identified the teHsp90 interactome in KSHV(+) primary effusion lymphoma cells. Viral and cellular proteins were identified, including many involved in nuclear factor (NF)-κB signaling, apoptosis, and autophagy. KSHV vFLIP is a viral oncoprotein homologous to cFLIPs, with NF-κB-activating and antiapoptotic activities. We show that teHsp90 binds vFLIP but not cFLIPs. Treatment with PU-H71 induced degradation of vFLIP and IKKγ, NF-κB downregulation, apoptosis and autophagy in vitro, and more importantly, tumor responses in mice. Analysis of the interactome revealed apoptosis as a central pathway; therefore, we tested a BCL2 family inhibitor in primary effusion lymphoma cells. We found strong activity and synergy with PU-H71. Our findings demonstrate PU-H71 affinity capture identifies actionable networks that may help design rational combinations of effective therapies.

Abstract 4829: A purine scaffold Hsp90 inhibitor has antitumor activity in KSHV-associated malignancies by suppressing vFLIP

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Background: Kaposis sarcoma herpesvirus (KSHV/ HHV-8), a member of the α-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposis sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which KSHV-infected PEL cells are protected from apoptosis. KSHV vFLIP has been identified as a viral oncogene that is responsible for NF-κB-dependent anti-apoptotic gene expression in PEL cells, and also prevents autophagy in KSHV-infected endothelial cells. In particular, the IKK signaling complex consisting of IKKα, IKKα, IKKα, vFLIP and Hsp90 was shown to be essential for survival in PEL cells. The chaperone protein Hsp90 binds client proteins involved in the regulation of cell survival and apoptosis signal transduction, including Akt, IKK complex, and KSHV vFLIP. A lack of Hsp90 causes protein misfolding, ubiquitination and degradation. The Hsp90 inhibitor geldanamycin was previously tested in PEL cells and shown to inhibit activity of the IKK complex in vitro. However, geldanamycin is of limited therapeutic potential due to its undesirable pharmacophysiology. We tested a new purine-scaffold Hsp90 inhibitor with high selectivity for tumor versus normal cell Hsp90, which is water-soluble with high oral bioavailability and excellent therapeutic window. Materials and methods: We evaluated the sensitivity of several KSHV infected and uninfected cell lines to treatment with this inhibitor, designated PU-H71. We performed viability and NF-κB reporter luciferase assays, and immunoblot and qRT-PCR analyses of cellular and viral proteins. Finally, we used a mouse PEL xenograft model and in vivo imaging to assess tumor responses to PU-H71. Results: We found all KSHV-positive PEL cell lines to be exquisitely sensitive when compared to uninfected lymphoma cells, with LC50s in the nanomolar range. PU-H71 was shown to induce PEL cell death by apoptosis and autophagy within 48 hours of treatment. PU-H71 also showed preferential toxicity in an in vitro model of KS. Pulldown demonstrated association of the compound with the active vFLIP-IKKα signalosome. Western blot analysis indicated that the IKK signaling complex was destabilized, and components vFLIP and IKKα were degraded upon PU-H71 treatment, resulting in inhibition of NF-κB signaling, as confirmed by reporter luciferase assay. qRT-PCR and reporter analysis indicated low levels of concomitant lytic reactivation. PU-H71 was further shown to inhibit progression of tumor spread and confer a significant survival advantage (p<0.02) in a mouse xenograft model of PEL, with persistence of IKKα destabilization ex vivo. Conclusions: Our findings demonstrate that Hsp90 inhibition with PU-H71 leads to reduced vFLIP levels in KSHV-infected cells, and to tumor responses. Thus, PU-H71 is a promising targeted approach for the treatment of KS and PEL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4829. doi:1538-7445.AM2012-4829

Identification of a nucleoside analog active against adenosine kinase–expressing plasma cell malignancies

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.

Abstract P3-04-08: The role of HER2 mutations in resistance to endocrine therapy in ER+ breast cancer

Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. Through whole-exome sequencing of metastatic tumor biopsies from patients with endocrine resistant ER+ metastatic breast cancer, we identified 13 different HER2 mutations, including five in the kinase domain, four in the signaling domain, three in the extracellular domain, and one in the transmembrane region of the protein. Two of the kinase domain mutations (L755S and V777L) have been previously described and shown to be activating and resistant to reversible anti-HER2 targeted therapies; the remaining mutations have not been reported. In several of these patients, whole exome sequencing of a pre-treatment primary tumor did not identify the HER2 mutations seen in the corresponding metastatic tumor, suggesting that they were acquired during therapy. To examine the role of HER2 mutations in endocrine resistance, we generated ER+ breast cancer cell lines (MCF7 and T47D) stably expressing the HER2 mutants observed in our clinical data. Several mutants promoted enhanced growth in charcoal dextran-stripped media, which lacks estradiol and mimics treatment with aromatase inhibitor. In addition, several mutants conferred varying degrees of resistance to fulvestrant and tamoxifen. Taken together, these results suggest that HER2 mutations are associated with acquired resistance to endocrine therapies in patients with ER+ breast cancer. The ability of irreversible anti-HER2 agents as well as other agents that target the HER2 pathway to overcome this resistance is being tested for individual HER2 mutations in vitro . The results from these studies may provide a clinical rationale for therapeutic combination strategies in patients with refractory tumors that have acquired endocrine resistance through HER2 mutations. Citation Format: Nayar U, Cohen O, Oh C, Wagle N. The role of HER2 mutations in resistance to endocrine therapy in ER+ breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-04-08.

Abstract 5110: A novel nucleoside analog therapeutically active against plasma cell malignancies and other ADK-expressing cancers including colon and pancreatic adenocarcinomas

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

Hsp90 is a viable therapeutic target in the treatment of KSHV-associated primary effusion lymphoma

BackgroundHsp90 is a chaperone protein that binds client proteinsinvolved in the regulation of cell survival and apoptosissignal transduction, including Akt, IKK complex, Apaf-1, survivin, CDKs, and KSHVvFLIP.Thisbindingisnecessary to maintain proper protein folding, assembly,transport, and function. A lack of Hsp90 results inprotein misfolding, ubiquitination, and degradation.KSHV vFLIP was identified as a viral gene that isresponsible for NF- B-dependent anti-apoptotic geneexpression in primary effusion lymphoma (PEL) cells[1]. In particular, the IKK signaling complex consistingof IKKa,IKKb,IKKg,vFLIP,andHsp90inthecaseofPEL cells was demonstrated as essential for survival inthese cells. As Hsp90 forms part of the IKK signalo-some in PEL cells, the Hsp90 inhibitor geldanamycinwas previously tested in these cells and has beenshown to inhibit activity of this complex in vitro. How-ever, geldanamycin is of limited therapeutic potentialdue to its undesirable pharmacophysiology. We testeda new purine-scaffold Hsp90 inhibitor with high selec-tivity for tumor versus normal cell Hsp90, which iswater-soluble with high oral bioavailability and excel-lent therapeutic window [2,3].Materials and methodsWe evaluated the sensitivity of several KSHV infectedand uninfected cell lines to treatment with this inhibi-tor, called PU-H71. We performed viability and NF- Breporter luciferase assays. Immunoblot analyses to cel-lularandviralproteinsweredonetoassesstheeffectof PU-H71. Finally, we used a mouse PEL xenograftmodel and in vivo imaging to assess tumor responsesto PU-H71.ResultsWe found all KSHV-positive PEL cell lines to be exqui-sitely sensitive when compared to uninfected lymphomacells, with growth inhibition at IC