Abstract: Exogenous cDNA introduced into an experimental system, either intentionally or accidentally, can appear as added read coverage over that gene in next-generation sequencing libraries derived from this system. If not properly recognized and managed, this cross-contamination with exogenous signal can lead to incorrect interpretation of research results. Yet, this problem is not routinely addressed in current sequence processing pipelines. Results We present cDNA-detector, a computational tool to identify and remove exogenous cDNA contamination in DNA sequencing experiments. We demonstrate that cDNA-detector can identify cDNAs quickly and accurately from alignment files. A source inference step attempts to separate endogenous cDNAs (retrocopied genes) from potential cloned, exogenous cDNAs. cDNA-detector provides a mechanism to decontaminate the alignment from detected cDNAs. Simulation studies show that cDNA-detector is highly sensitive and specific, outperforming existing tools. We apply cDNA-detector to several highly-cited public databases (TCGA, ENCODE, NCBI SRA) and show that contaminant genes appear in sequencing experiments where they lead to incorrect coverage peak calls. Conclusions cDNA-detector is a user-friendly and accurate tool to detect and remove cDNA detection in NGS libraries. This two-step design reduces the risk of true variant removal since it allows for manual review of candidates. We find that contamination with intentionally and accidentally introduced cDNAs is an underappreciated problem even in widely-used consortium datasets, where it can lead to spurious results. Our findings highlight the importance of sensitive detection and removal of contaminant cDNA from NGS libraries before downstream analysis.

Abstract: For patients with hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) metastatic breast cancer (MBC), first-line treatment is endocrine therapy (ET) plus cyclin-dependent kinase 4/6 inhibition (CDK4/6i). After disease progression, which often comes with ESR1 resistance mutations (ESR1-MUT), which therapies to use next and for which patients are open questions. An active area of exploration is treatment with further CDK4/6i, particularly abemaciclib, which has distinct pharmacokinetic and pharmacodynamic properties compared with the other approved CDK4/6 inhibitors, palbociclib and ribociclib. We investigated a gene panel to prognosticate abemaciclib susceptibility in patients with ESR1-MUT MBC after palbociclib progression. METHODS We examined a multicenter retrospective cohort of patients with ESR1-MUT MBC who received abemaciclib after disease progression on ET plus palbociclib. We generated a panel of CDK4/6i resistance genes and compared abemaciclib progression-free survival (PFS) in patients without versus with mutations in this panel (CDKi-R[–] v CDKi-R[+] ). We studied how ESR1-MUT and CDKi-R mutations affect abemaciclib sensitivity of immortalized breast cancer cells and patient-derived circulating tumor cell lines in culture. RESULTS In ESR1-MUT MBC with disease progression on ET plus palbociclib, the median PFS was 7.0 months for CDKi-R(–) (n = 17) versus 3.5 months for CDKi-R(+) (n = 11), with a hazard ratio of 2.8 ( P = .03). In vitro, CDKi-R alterations but not ESR1-MUT induced abemaciclib resistance in immortalized breast cancer cells and were associated with resistance in circulating tumor cells. CONCLUSION For ESR1-MUT MBC with resistance to ET and palbociclib, PFS on abemaciclib is longer for patients with CDKi-R(–) than CDKi-R(+). Although a small and retrospective data set, this is the first demonstration of a genomic panel associated with abemaciclib sensitivity in the postpalbociclib setting. Future directions include testing and improving this panel in additional data sets, to guide therapy selection for patients with HR+/HER2– MBC.

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

Abstract: Purpose: Resistance to endocrine therapies in estrogen receptor positive (ER+) metastatic breast cancer (MBC) is widespread, and understanding the mechanisms whereby these tumors acquire resistance is a critical need. We and others previously described acquired activating hotspot HER2 (ERBB2) mutations in ~5% of ER+ MBC that conferred resistance to multiple ER-targeting therapies, including the selective estrogen receptor degrader fulvestrant. These tumors could be re-sensitized to fulvestrant in vitro through addition of the irreversible pan-HER tyrosine kinase inhibitor neratinib, suggesting a possible clinical combination strategy for patients. Although some HER2 mutations are relatively more frequent in tumors, there is a “long tail” of rare HER2 mutations that have not been characterized but remain clinically important for patients whose tumors harbor them. Therefore, there is biological and clinical value in prospectively characterizing all possible missense mutations in HER2. Methodology: Since only activating HER2 mutations conferred resistance to fulvestrant (and not passenger or inactivating mutations), resistance to fulvestrant in ER+ breast cancer cells can be used as a surrogate kinase assay for HER2 activity. Therefore, we are performing a saturation mutagenesis screen of HER2, using fulvestrant resistance as a readout for activating mutants. Screen optimization included: a) testing selected mutants cloned into a custom vector to identify appropriate positive and negative controls for screen QC, b) custom screen design (transduction under ER inhibition, and an empirically-determined ratio of growth in fulvestrant vs DMSO) to enable recovery of mutants of varying growth phenotypes. We also designed PCR conditions to enable efficient amplification of HER2 (the largest ORF ever tested by saturation mutagenesis) from genomic DNA, and custom-designed and built a comprehensive HER2 library that includes built-in controls such as stop codons. The saturation mutagenesis screen is currently underway, and will generate putative activating HER2 mutations (i.e., not growth-inhibited in fulvestrant versus complete media), that will be validated in a “minipool” screen. Validated hits will be further tested for sensitivity to the combination of fulvestrant+neratinib or other kinase inhibitors. Summary and conclusions: We have designed a saturation mutagenesis screen to recover a spectrum of activating HER2 mutations, the largest such library generated to date, and using resistance to the ER inhibitor fulvestrant as a novel surrogate kinase assay. This screen will generate a comprehensive reference table of HER2 mutant phenotypes in terms of response and resistance to ER and HER2 targeting agents. These findings will have translational applicability, and may suggest promising precision medicine approaches for clinical management of patients harboring somatic HER2 mutations. Citation Format: Utthara Nayar, Federica Piccioni, Xiaoping Yang, David Root, J T. Neal, Lisa D. Eli, Irmina Diala, Alshad S. Lalani, Nikhil Wagle. Phenotypic characterization of a comprehensive set of HER2 missense mutants in ER+ breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1914.

Abstract: While recent studies have begun characterizing the metastatic breast cancer (MBC) genomics, our understanding of mechanisms of acquired endocrine-resistance, their induced cell-state, and their altered drug-response profile, remains lacking. We collected biopsies from patients with MBC with detailed clinicopathologic features. To date we profiled 520 exomes, and 291 transcriptomes, with 126 patients having multiple biopsy exomes. Curated endocrine-relapse set include 60 patients with pre-treatment and post-relapse exomes. Characterization of candidate mechanisms of resistance (MOR) included 909 RNA-seq profiles of T47D cells with introduced MOR under various drugs. In the acquired endocrine resistance cohort, as expected, we found frequent ESR1 acquired mutations (13 pt, 22%). Additionally, we identified acquired activating SNVs and amplifications in oncogenic receptor tyrosine kinases (RTKs) in 18/60 (30%), including EGF family - HER2 (n=7), ERRB3 (R525Q), and EGFR (S116F), and FGF family - FGFR1 (n=5), FGFR2 (n=4), and FGF3 amplicon (n=4). RNA-seq of T47D cells overexpressing HER2 activating mutations revealed a distinct cell-state (HER2-ACT). Similarly, FGFR activation revealed a district FGFR-ACT state. The transcriptional signatures of these HER2 and FGFR states were remarkably similar (odd-ratio= 91, p= 2.64E-94). To characterize the cell-state common to HER and FGFR, we defined RTK-ACT with 358 overlapping marker genes (see table). Canonical (estradiol) ER signaling is slightly elevated in RTK-ACT, however this state is strongly associated with growth-factor driven ER signaling, suggesting reprogramming of ER from AF2, to AF1 signaling. Consistent with this, RTK-ACT had significantly higher MAPK activation. Additionally, RTK-ACT Induced stronger similarity to Basal-state, enrichment in motility/migration, mesenchymal, and stem-like features, with top genes including CDH3, MBP7, and S100, ETV, DUSP, SPRY families (see table). To study RTK-driven state in-vivo, we analyzed RNA-seq from our MBC biopsies, and compared tumors with activating RTK mutations (n=38) with WT (n=118), and inferred activated RTK in-vivo (RTK.ACT.iv). Our in-vitro and in-vivo states show significant overlap (OR=4.71, p=9.42E-20). Furthermore, characterization of RTK.ACT.iv, recapitulated all the cell-state features observed in RTK.ACT - including higher growth-factors ER signaling, MAPK, and basal-like state (see table). We further studied the viability and transcription of these RTKs under various drugs (12 treatments), including fulvestrant, palbociclib, and specific tyrosine kinase inhibitors (TKIs) - Neratinib (pan-HER inhibitor) and FIIN-3 (FGFR-i). We found that HER2-ACT and FGFR-ACT signatures remain robust when treated with fulvestrant, palbociclib, and their combinations, as compared to TKIs suppression (see table). in-line with our viability results - suggesting intrinsic resistance to CDK4/6i and sensitivity to TKIs. This study demonstrated that activating RTKs constitute of prevalent modality of acquired resistance to endocrine therapies, inducing a distinct state with clinical implications - suggesting the potential benefit of combination therapies with specific TKIs over CDK4/6i. The common MAPK activity and our preliminary results - suggests the potential of convergence-node targeting strategy with added MEK or SHP2 inhibition. TableGene set AGene set BOdds-ratioP-value (two-sided)Fisher''s Exact test matHER2-ACT -FGFR-ACT -912.64E-9469, 131, 131, 22704HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC (top 200)FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC (top 200)HER2-ACT_1000 -FGFR-ACT_1000 -18.98.28E-248358, 642, 642, 21758HER2 S653C, L755S, V777L, L869R vs. GFP, under DMSO, rank genes based on the logFC- top 1000FGFR1, FGFR2 (WT, N550K, M538I, K660N) vs. GFP, Parental, Under DMSO, rank genes based on the logFC - top 1000RTK-ACTHALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB3.030.004229, 191, 349, 22474RTK-ACTER driven by Growth Factors, PMID: 208897186.771.74E-059, 86, 349, 22585RTK-ACTMEK_UP.V1_UP (MAPK), MSigDB10.95.72E-1827, 169, 331, 22496RTK-ACTRAS ONCOGENE (MAPK), PMID: 162730928.445.77E-1220, 158, 338, 22553RTK-ACTWU_CELL_MIGRATION, PMID 187243907.648.96E-1119, 165, 339, 22502RTK-ACTHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 17409405131.38E-079, 45, 349, 22621RTK-ACTHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB3.770.00031411, 189, 347, 22477RTK-ACTLIM_MAMMARY_STEM_CELL_UP, PMID 203461513.119.56E-0622, 467, 336, 22205RTK-ACTRTK-ACT.iv -4.719.42E-2060, 940, 298, 21992In MBC biopsies, compare RTK mutations with WT, rank genes based on the logFC - top 1000RTK-ACT.iv (in-vivo)HALLMARK_ESTROGEN_RESPONSE_EARLY, MSigDB1.950.020316, 184, 984, 22088RTK-ACT.ivER driven by Growth Factors, PMID: 208897182.640.0076310, 85, 990, 22193RTK-ACT.ivMEK_UP.V1_UP (MAPK), MSigDB2.863.91E-0522, 174, 978, 22098RTK-ACT.ivRAS ONCOGENE (MAPK), PMID: 162730921.620.13212, 166, 988, 22152RTK-ACT.ivWU_CELL_MIGRATION, PMID 187243903.573.60E-0725, 159, 975, 22115RTK-ACT.ivHUPER_BREAST_BASAL_VS_LUMINAL_UP, PMID 174094059.515.43E-1016, 38, 984, 22235RTK-ACT.ivHALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, MSigDB2.090.0073817, 183, 983, 22090RTK-ACT.ivLIM_MAMMARY_STEM_CELL_UP, PMID 203461511.420.089229, 460, 971, 21819HER2-ACTHER2-ACT - Fulv3006.53E-183109, 91, 91, 22750HER2-ACTHER2-ACT - Palbo2331.46E-163101, 99, 99, 22749HER2-ACTHER2.ACT - Fulv + Palbo2197.64E-15999, 101, 101, 22742HER2-ACTHER2.ACT - Neratinib63.19.78E-7257, 143, 143, 22707HER2-ACTHER2.ACT - Fulv + Neratinib59.33.53E-6855, 145, 145, 22724FGFR-ACTFGFR.ACT - Palbo19003.2e-319157, 43, 43, 22778FGFR-ACTFGFR.ACT - Fulv + Palbo12501.38E-290148, 52, 52, 22767FGFR-ACTFGFR.ACT - Fulv8661.72E-266140, 60, 60, 22764FGFR-ACTFGFR.ACT - Palbo + FIIN.31062.88E-10474, 126, 126, 22710FGFR-ACTFGFR.ACT - Fulv + Palbo + FIIN.378.41.14E-8464, 136, 136, 22705FGFR-ACTFGFR.ACT - FIIN.367.42.16E-7559, 141, 141, 22730FGFR-ACTFGFR.ACT - Fulv + FIIN.336.89.47E-4541, 159, 159, 22733 Citation Format: Ofir Cohen, Pingping Mao, Utthara Nayar, Jorge E Buendia-Bue, Dewey Kim, Esha Jain, Karla Helvie, Daniel Abravanel, Kailey J Kowalski, Christian Kapstad, Samuel Freeman, Victor Adalsteinsson, Seth A Wander, Adrienne G Waks, Gad Getz, Aviv Regev, Eric Winer P Winer, Nancy U Nancy U. Lin, Nikhil Wagle. Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-02.

Abstract: Primary effusion lymphoma (PEL) is a largely incurable malignancy of B cell origin, driven by infection with KSHV/HHV-8, and with notable plasmacytic differentiation. PEL carries an extremely poor prognosis with a median survival time of less than 6 months and is known to be largely resistant to conventional chemotherapy. Therefore, we conducted high throughput screens for effective inhibitors using PEL cell lines. We discovered a compound, 6-ethylthioinosine (6-ETI), a nucleoside analog with selective toxicity to PEL and multiple myeloma (MM) at nanomolar concentrations, but not to other lymphoma cell lines tested. 6-ETI also showed remarkable responses in a mouse xenograft model of PEL. To understand the molecular mechanism(s) of selectivity towards PEL, we developed and performed resistome analysis, an unbiased approach based on RNA sequencing of in vitro and in vivo generated resistant subclones. We found adenosine kinase (ADK) inactivating genomic alterations in all resistant clones as the mechanism of resistance. Concordantly, we found that plasma cells express ADK, as well as sensitive PELs and multiple myeloma cell lines, while resistant lymphoma cell lines including those with EBV infection had lower levels (if any); interestingly, the latter could be sensitized by cell crowding-induced ADK upregulation. Like other nucleoside analogues, 6-ETI induces ATP-depletion and cell death accompanied by S-phase arrest and DNA damage, but only in ADK-expressing cells. Immunohistochemistry for ADK served as a new biomarker approach to identify tumors that may be sensitive to 6-ETI, which we documented for primary specimens of PEL, multiple myeloma and other lymphoid malignancies with plasmacytic features, namely plasmablastic lymphoma. A number of nucleoside analogues have been reported to be effective in treating leukemias and B and T cell lymphomas. We performed structure-activity relationship studies and tested a number of nucleoside analogs that are in preclinical or clinical development for other hematological malignancies to identify and better understand the structural requirements for 6-ETIÕs biological activity. We successfully demonstrated that 6-ETI was more potent and selective at killing PEL and MM cell lines than other studied nucleoside analogs suggesting that this compound possesses unique and distinct features that are clinically promising. Despite the presence of treatment approaches that can greatly extended the survival of MM patients, resistance remains an issue, warranting the need for new effective agents and combinations. We identified 6-ETI as a novel and clinically viable nucleoside analog for the treatment of PEL, immunoblastic lymphoma, plasmablastic lymphoma, multiple myeloma and other ADK-expressing cancers. Figure 1. Expression of ADK and sensitivity to 6-ETI in plasma cell tumors. (A) BC3 cells ADK expression was evaluated by immunohistochemistry in the BC3 cell line, hyperplastic tonsils and PEL, multiple myeloma and plasmablastic lymphoma primary tumors. 60X original magnification is shown. In the image of a tonsil section, a positive cell with morphological features of a plasma cell is enlarged in the insert. Original magnification 60X. (B) LC50s for multiple myeloma cell lines treated with 6-ETI were determined by CellTiter-Glo assay. BC3 was used as a positive control and IBL1 as a negative control for drug sensitivity. Shown are the average of two independent experiments, where each condition was performed in duplicate in each experiment. (C) Model for 6-ETIÕs mechanism of action within the cell is illustrated, where 6-ETI competes with adenosine (ADO) and other nucleosides for binding to and phosphorylation by ADK, which inhibits ATP-dependent metabolic processes. This also allows 6-ETI to be phosphorylated and activated by ADK, with subsequent phosphorylation steps that allow the compound to be incorporated into DNA and possibly RNA, leading to DNA synthesis inhibition, DNA damage response, and cell death. Figure 1. Expression of ADK and sensitivity to 6-ETI in plasma cell tumors. (A) BC3 cells ADK expression was evaluated by immunohistochemistry in the BC3 cell line, hyperplastic tonsils and PEL, multiple myeloma and plasmablastic lymphoma primary tumors. 60X original magnification is shown. In the image of a tonsil section, a positive cell with morphological features of a plasma cell is enlarged in the insert. Original magnification 60X. (B) LC50s for multiple myeloma cell lines treated with 6-ETI were determined by CellTiter-Glo assay. BC3 was used as a positive control and IBL1 as a negative control for drug sensitivity. Shown are the average of two independent experiments, where each condition was performed in duplicate in each experiment. (C) Model for 6-ETIÕs mechanism of action within the cell is illustrated, where 6-ETI competes with adenosine (ADO) and other nucleosides for binding to and phosphorylation by ADK, which inhibits ATP-dependent metabolic processes. This also allows 6-ETI to be phosphorylated and activated by ADK, with subsequent phosphorylation steps that allow the compound to be incorporated into DNA and possibly RNA, leading to DNA synthesis inhibition, DNA damage response, and cell death. Disclosures Nayar: Weill Cornell Medical College: Patents & Royalties: Submitted patent applicatio for 6-ETI. Cesarman:Weill Cornell Medical College: Patents & Royalties: applied for patent for 6-ETI.

Abstract: Abstract 160 Kaposi's sarcoma herpesvirus (KSHV/HHV-8), a member of the γ-herpesvirus family of human DNA viruses, is the etiologic agent of several malignancies in immune-compromised individuals, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Activated NF-κB is a critical mechanism by which lymphoma cells infected by KSHV are protected from apoptotic stress. vFLIP is the viral protein largely responsible for the constitutive NF-κB activity, and is essential for PEL cell survival. Using a subclone of the BC3 PEL cell line that stably expresses firefly luciferase under the control of an NF-κB promoter (BC3/NFκB-luc), we performed high throughput screening (HTS) of compound libraries to identify inhibitors of vFLIP/NF-κB in PEL cells. Assay optimization was done using the NCI Training set, following which screening of the NIH diversity set at 5μM picked up 60 hits (with 〉 50% inhibition) from a total of ∼2000 compounds. To further test these compounds, we introduced a renilla luciferase controlled by a constitutive promoter (retroviral LTR) to obtain a double reporter cell line (BC3-REN#3). Three compounds showed preferential inhibition of firefly luciferase in the double-reporter cell line, and/or preferential inhibition of the BC3/NFκB-luc cell line when compared to a control non-lymphoid cell line with constitutive luciferase expression (U251-pGL3). Analogs of these three compounds were further tested in BC3-REN#3 cells, and three analogs were identified with increased activity/specificity for NF-κB. One of the compounds identified so far, here designated NSC-E, has shown dose-dependent inhibition of NF-κB in PEL cells, as evaluated independently by reporter and electrophoretic mobility shift assays (EMSA). Nuclear translocation of the NF-κB active subunit p65, but not inactive p50 is inhibited by this compound, although it does not alter upstream events in the NF-κB pathway, such as IKKα/β phosphorylation or IκBα degradation. Studies are ongoing to elucidate the direct molecular target of NSC-E. Inhibition of NF-κB by NSC-E occurs only in PEL cell lines, and not in other lymphoma cell types. In addition, this compound preferentially inhibits viability of KSHV-infected lymphoma cells, at LC50s ranging from 18nM to ∼200nM in various PEL cell lines tested. In contrast, an effect on EBV-infected lymphoma cell viability is only apparent at much higher doses (LC50s in the 20–80μM range). Hence, ∼300X the KSHV-effective dose of NSC-E would be required to achieve inhibition in EBV-associated or virus-negative lymphomas, suggesting this inhibitor is very KSHV-specific. Finally, NSC-E was evaluated in a traceable reporter xenograft mouse model of KSHV-associated PEL using in vivo imaging, and found to be highly efficacious against both incipient and well-established tumors, with 100% tumor responses and no apparent toxicity. Thus, we have identified a novel, specific, and highly effective inhibitor of KSHV-associated lymphoma that demonstrates clear pre-clinical promise for therapeutic applications. Disclosures: No relevant conflicts of interest to declare.