Hippuristanol Synthesis Essay

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  • Targeting eIF4A chemosensitizes Myc-driven tumors to DNA damaging agents

    We took advantage of the Eμ-Myc model to assess the in vivo chemosensitization potential of Hipp using the approach outlined in Figure 1a. This model has been used to identify and characterize novel oncogenes and tumor suppressor pathways23 for testing the contribution of effector pathways to tumor initiation24 and maintenance,5 and for assessing response to chemotherapy.5, 24 We took advantage of engineered Tsc2+/−-Myc and Pten+/−-Myc tumors as these are dependent on Mcl-1 for their survival,3 exhibit deregulated mTORC1 activity—a central regulator of translation initiation, and are genes for which mutations have been documented in human Burkitt’s lymphoma.25 Treatment of mice bearing Tsc2+/−-Myc lymphomas with Hipp did not induce any remissions at the doses tested, whereas doxorubicin (Dxr) or rapamycin (Rap) induce short-lived remissions (Figure 1b and Supplementary Figure S1A).3 Hipp however synergized with Dxr in vivo to extend tumor-free survival up to 17 days (Figure 1b; P<0.001 for Hipp+Dxr versus Dxr). Treatment of mice with Hipp lead to an in vivo reduction in protein synthesis in tumor cells, as assessed by a reduction in polysome/monosome ratio in tumor cells isolated from Hipp-treated mice (Figure 1c) and in vivo monitoring of protein synthesis in Tsc2+/−-Myc tumor cells using SUnSET (Figure 1d).26 Likewise, exposing Tsc2+/−-Myc lymphoma cells to 50 nM Hipp led to a similar reduction in protein synthesis (Supplementary Figure S1B)—an effect that was not due to loss of cell viability (Supplementary Figure S1C). We observed an increase in apoptosis associated with Hipp’s ability to alter the Dxr-response in Tsc2+/−-Myc tumors, as judged by TUNEL staining of tumor tissue (Figures 1e and f, and Supplementary Figure S1D). No apparent difference in Ki-67 staining was detected among samples from Hipp-, Dxr- or Hipp+Dxr-treated mice (Supplementary Figure S1E).

    We then sought to determine whether the results described above for the Tsc2+/−-Myc setting could be extended to different tumor genotypes and chemotherapeutic agents. We found that Hipp and Dxr also synergized in animals bearing Pten+/−-Myc (Figure 2a) or Eμ-Myc/eIF4E tumors (Figure 2b). The latter result is particularly noteworthy, as eIF4E is a genetic modifier of the Rap-response5 and elevated eIF4E levels lead to resistance to Rap+Dxr combination treatment (Figure 2b). These experiments extend the utility of Hipp in chemosensitizing cells to include tumors with lesions upstream of Tsc1/2 and downstream of mTOR. Hipp also synergized with cyclophosphamide (CTX), a standard-of-care chemotherapeutic used in the treatment of lymphomas (Figure 2c). These results indicate that Hipp is effective at resensitizing Myc-driven lymphomas to DNA damaging agents in vivo.

    To address concerns regarding toxicity in vivo, we administered Hipp to mice at the effective doses and monitored body weight and liver function (Supplementary Figures S2A, B). Treated animals neither suffered from weight loss (Supplementary Figure S2A) nor showed any signs of liver cell damage as assessed by ALT and AST levels (Supplementary Figure S2B). Among the hematological parameters that we measured, there was little difference in B220+ (B-cell), Ly-6G+ (granulocytes), CD11b+ (monocyte/macrophages, granulocytes) and CD4+ (T cell) populations when comparing vehicle to Hipp-treated mice (Supplementary Figure S2C). These results are similar to what has been reported for silvestrol, a potent rocaglamide that interferes with eIF4A activity.9 Taken together, they indicate that transient inhibition of eIF4A is well tolerated at the organismal level. We also tested whether Hipp was a substrate for Pgp-1 (MDR1), a major drug efflux protein implicated in chemoresistance.27 A significant increase in IC50 of Dxr or silvestrol was noted in cells expressing high levels of Pgp-1 (Supplementary Figures S2D, E), consistent with these being substrates for Pgp-1.18 This phenomenon was not observed with Hipp indicating that it is not a Pgp-1 substrate (Supplementary Figure S2F).

    Mcl-1 and Bcl-2 are genetic modifiers of the Hipp+Dxr synergy response in Eμ-Myc lymphomas

    Mcl-1 and Bcl-2 are known modifiers of drug sensitivity. To determine if altering Mcl-1 or Bcl-2 levels could affect the Hipp+Dxr synergy response, we generated Eμ-Myc lymphomas overexpressing these anti-apoptotic proteins (Figure 3a, lanes 2 and 3). Mice bearing these lymphomas displayed a poor response to Hipp+Dxr and Rap+Dxr combination treatments (Figures 3b and c). To extend these results, we tested whether the pro-apoptotic ‘BH3-only’ family members, Bim (which interacts with Bcl-2, Bcl-XL, Bcl-W, and Mcl-1) and Noxa (which interacts with Mcl-1) could also modulate the tumor response to Hipp/Dxr. Two shRNAs for each target were developed and caused a greater than four-fold reduction in Bim protein (Figure 3d) and Noxa mRNA (Figure 3e) levels. (Note that we could not probe for NOXA protein levels due to poor reactivity of available antibodies.) Infection of Tsc2+/−-Myc tumor cells with either Bim or Noxa shRNAs significantly dampened the in vivo ability of Hipp and Dxr to synergize (Figures 3f and g). To determine if these effects were due to altered sensitivity to Hipp and/or Dxr, Tsc2+/−-Myc lymphomas were exposed ex vivo to single agents and cell viability was assessed. A nearly two- and five- fold increase in the resistance to Hipp and Dxr, respectively, was observed upon ectopic overexpression of Mcl-1 (Supplementary Figures S3A, B). A smaller but reproducible increase in the resistance to Hipp and Dxr was noted upon Bim and Noxa suppression in Tsc2+/−-Myc lymphomas (Supplementary Figures S3C–F). (These experiments could not be performed with Eμ-Myc/Bcl-2 tumor cells since we are unable to propagate these ex vivo.) Although these results do not rule out a contribution from tumor cell extrinsic responses to the Hipp+Dxr-response in vivo, they do indicate that components of the intrinsic cell death pathway are major modifiers of the Hipp+Dxr synergy response in vivo and do so at least in part by affecting cell sensitivity to Hipp and Dxr.

    Hipp sensitizes Myc-driven tumors to Bcl-2-targeted therapeutics

    The effectiveness of Hipp in reducing Mcl-1 levels (see below) and the role that Bcl-2 family members have in mediating resistance to Hipp’s chemosensitizing properties (Figure 3) suggested that a combination of Hipp and Bcl-2 targeted therapy might be an appropriate strategy to curtail chemoresistance arising from the activation of the intrinsic cell death pathway. To test this, we generated a series of isogenic lines ectopically expressing Mcl-1, Bcl-2 or both, using Arf−/−-Myc and p53−/−-Myc cells (Figure 4a, Supplementary Figures S4–S6). Ectopic expression of Mcl-1 or Bcl-2 in Arf−/−Eμ-Myc or p53−/−-Myc cells produced lines that displayed an increase in the Hipp IC50, with Bcl-2 expressing cells demonstrating a more significant shift than cells expressing Mcl-1 (Figure 4b and Supplementary Figure S4). Expression of Bcl-2 in Arf−/−-Myc or p53−/−-Myc cells sensitized these to ABT-737 (Supplementary Figure S4)—an expected phenomenon that is thought to be the consequence of elevated BH3 protein levels predisposing to Bcl-2 dependence and response to ABT-737.28 No synergy was observed between Hipp and ABT-737 in Arf−/−-Myc/Mcl-1 or p53−/−-Myc/Mcl-1 cells (Supplementary Figures S5A and S5C). Arf−/−-Myc/Bcl-2 and p53−/−-Myc/Bcl-2 showed synergy at concentrations of Hipp >160 nM (Supplementary Figures S5B, S5D–S5F). Although there was little synergy between Hipp and ABT-737 in either Arf−/−-Myc or p53−/−-Myc parental cell lines, ectopic expression of Bcl-2 and Mcl-1 led to a strong synergistic relationship between Hipp and ABT-737 (Figure 4c and Supplementary Figure S6). Mcl-1 levels in Arf−/−-Myc/Mcl-1/Bcl-2 cells are significantly depleted upon exposure of cells to Hipp (Figure 4d).

    Suppression of eIF4AI is sufficient to chemosensitize Myc-driven tumors to Bcl-2 targeted therapeutics

    Mammalian cells encode for two eIF4A isoforms, eIF4AI and eIF4AII, that share 90% similarity at the protein level and have non-redundant but overlapping activities.29, 30 As Hipp inhibits both isoforms, we sought to determine if RNAi-mediated suppression of either or both isoform could phenocopy the ABT-737 sensitization. Long-term (6 days) suppression of eIF4AI, but not eIF4AII, was lethal (Figures 5a and b), with little change in the percentage of viable uninfected cells (Supplementary Figure S7A). This effect was p53-dependent, as it was significantly blunted in p53−/−-Myc/Bcl-2 cells (Supplementary Figure S7B). Suppression of eIF4AII, eIF4E or Mcl-1, as well as expression of a neutral shRNA targeting Firefly luciferase (shFLuc) had a minor impact on the GFP+ cell population (∼20% change) (Figure 5b, Supplementary Figure S7B). These results indicate that long-term suppression of eIF4AI in Arf−/−-Myc/Bcl-2 cells is lethal.

    To assess if suppression of eIF4AI and/or eIF4AII would synergize with ABT-737 on a shorter time scale, we infected Arf−/−-Myc/Bcl-2 and p53−/−-Myc/Bcl-2 cells with retroviruses expressing shRNAs and exposed the cells to either vehicle or ABT-737 for a short-term pulse (that is, 18 hours) (Figure 5c, Supplementary Figure S7C). We then monitored the fitness of shRNA-expressing cells (GFP+) relative to uninfected cells (GFP) in a competition assay to detect altered sensitivity to ABT-737. The results demonstrate that ABT-737 had little effect on the viability of Arf−/−-Myc/Bcl-2 expressing shFLuc.1309, whereas loss of viability was apparent upon Mcl-1 suppression (Figure 5c, left panel; see shMcl-1.1334). Suppression of eIF4AI, but not eIF4AII, led to a dose-dependent loss in Arf−/−-Myc/Bcl-2 cell viability in the presence of ABT-737 (Figure 5c, right panel), which was significantly blunted upon loss of p53 (Supplementary Figure S7C, right panel). These results indicate that suppression of eIF4AI in Arf−/−-Myc/Bcl-2 cells is sufficient to synergize with ABT-737.

    Hipp and ABT-737 synergize in human lymphoma and leukemia tumor cells

    To extend our results to the human setting, we tested the sensitivity of a number of lymphoma and leukemic tumor cells to Hipp (Figure 6 and Supplementary Figure S8). Viability of the Burkitt’s lymphoma lines, Daudi, Ramos and Raji was reduced by Hipp (with IC50s ranging from 300 nM to 1.25 μM), whereas BJAB and Namalwa were relatively resistant with 80% of cells surviving at concentrations as high as 1.25 μM (Figure 6a). In addition, many other lymphoma/leukemic lines tested appeared relatively resistant to Hipp, with MV-4-11 and Mino being the more sensitive ones. Lines hMB, Granta 519 and Sc-1 showed less than a 10% reduction in viability when exposed to concentrations as high as 1250 nM (Supplementary Figure S8A). However, despite this, we found synergy between Hipp and ABT-737 in all Burkitt’s (Figure 6b) and lymphoma/leukemia lines (Supplementary Figure S8B) tested, although the extent varied among cell lines. In contrast, no synergy was observed between Hipp (5–625nM) and ABT-737 (156 nM–2.5 μM) in immortalized hTert-BJ cells (R Cencic, unpublished data), hinting that such activity may not extend to the non-transformed setting. No correlation was apparent between Hipp sensitivity (Figure 6a and Supplementary Figure S8A) and expression levels of eIF4A or PDCD4 (a repressor of eIF4A) (Figure 6c and Supplementary Figure S8C) in the cell lines tested. As well, Hipp+ABT-737 synergy appeared independent of p53 mutation status and levels of Bcl-2, Mcl-1, Bcl-XL or Bim (Figure 6c and Supplementary Figure S8C). These results indicate that ABT-737 and Hipp synergize in the majority of transformed lymphoma/leukemia lines tested.

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