Susan K. Peirce

Mitochondrial Bcl-2 family dynamics define therapy response and resistance in neuroblastoma

Neuroblastoma is a childhood tumor in which transient therapeutic responses are typically followed by recurrence with lethal chemoresistant disease. In this study, we characterized the apoptotic responses in diverse neuroblastomas using an unbiased mitochondrial functional assay. We defined the apoptotic set point of neuroblastomas using responses to distinct BH3 death domains providing a BH3 response profile and directly confirmed survival dependencies. We found that viable neuroblastoma cells and primary tumors are primed for death with tonic sequestration of Bim, a direct activator of apoptosis, by either Bcl-2 or Mcl-1, providing a survival dependency that predicts the activity of Bcl-2 antagonists. The Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 showed single-agent activity against only Bim:Bcl-2 primed tumor xenografts. Durable complete regressions were achieved in combination with noncurative chemotherapy even for highest risk molecular subtypes with MYCN amplification and activating ALK mutations. Furthermore, the use of unique isogenic cell lines from patients at diagnosis and at the time of relapse showed that therapy resistance was not mediated by upregulation of Bcl-2 homologues or loss of Bim priming, but by repressed Bak/Bax activation. Together, our findings provide a classification system that identifies tumors with clinical responses to Bcl-2 antagonists, defines Mcl-1 as the principal mediator of Bcl-2 antagonist resistance at diagnosis, and isolates the therapy resistant phenotype to the mitochondria.

PTEN and PI-3 kinase inhibitors control LPS signaling and the lymphoproliferative response in the CD19+ B cell compartment

Pattern recognition receptors (PRRs), e.g. toll receptors (TLRs) that bind ligands within the microbiome have been implicated in the pathogenesis of cancer. LPS is a ligand for two TLR family members, TLR4 and RP105 which mediate LPS signaling in B cell proliferation and migration. Although LPS/TLR/RP105 signaling is well-studied; our understanding of the underlying molecular mechanisms controlling these PRR signaling pathways remains incomplete. Previous studies have demonstrated a role for PTEN/PI-3K signaling in B cell selection and survival, however a role for PTEN/PI-3K in TLR4/RP105/LPS signaling in the B cell compartment has not been reported. Herein, we crossed a CD19cre and PTEN(fl/fl) mouse to generate a conditional PTEN knockout mouse in the CD19+ B cell compartment. These mice were further crossed with an IL-14α transgenic mouse to study the combined effect of PTEN deletion, PI-3K inhibition and expression of IL-14α (a cytokine originally identified as a B cell growth factor) in CD19+ B cell lymphoproliferation and response to LPS stimulation. Targeted deletion of PTEN and directed expression of IL-14α in the CD19+ B cell compartment (IL-14+PTEN-/-) lead to marked splenomegaly and altered spleen morphology at baseline due to expansion of marginal zone B cells, a phenotype that was exaggerated by treatment with the B cell mitogen and TLR4/RP105 ligand, LPS. Moreover, LPS stimulation of CD19+ cells isolated from these mice display increased proliferation, augmented AKT and NFκB activation as well as increased expression of c-myc and cyclinD1. Interestingly, treatment of LPS treated IL-14+PTEN-/- mice with a pan PI-3K inhibitor, SF1126, reduced splenomegaly, cell proliferation, c-myc and cyclin D1 expression in the CD19+ B cell compartment and normalized the splenic histopathologic architecture. These findings provide the direct evidence that PTEN and PI-3K inhibitors control TLR4/RP105/LPS signaling in the CD19+ B cell compartment and that pan PI-3 kinase inhibitors reverse the lymphoproliferative phenotype in vivo.

EGFR signaling defines Mcl-1 survival dependency in neuroblastoma

The pediatric solid tumor neuroblastoma (NB) often depends on the anti-apoptotic protein, Mcl-1, for survival through Mcl-1 sequestration of pro-apoptotic Bim. High affinity Mcl-1 inhibitors currently do not exist such that novel methods to inhibit Mcl-1 clinically are in high demand. Receptor tyrosine kinases (RTK) regulate Mcl-1 in many cancers and play a role in NB survival, yet how they regulate Bcl-2 family interactions in NB is unknown. We found that NB cell lines derived to resist the Bcl-2/-xl/-w antagonist, ABT-737, acquire a dependence on Mcl-1 and show increased expression and activation of the RTK, EGFR. Mcl-1 dependent NB cell lines derived at diagnosis and from the same tumor following relapse also have increased EGFR expression compared to those dependent on Bcl-2. Inhibition of EGFR by shRNA or erlotinib in Mcl-1 dependent NBs disrupts Bim binding to Mcl-1 and enhances its affinity for Bcl-2, restoring sensitivity to ABT-737 as well as cytotoxics in vitro. Mechanistically treatment of NBs with small molecule inhibitors of EGFR (erlotinib, cetuximab) and ERK (U0126) increases Noxa expression and dephosphorylates Bim to promote Bim binding to Bcl-2. Thus, EGFR regulates Mcl-1 dependence in high-risk NB via ERK-mediated phosphorylation of Bim such that EGFR/ERK inhibition renders Mcl-1 dependent tumors now reliant on Bcl-2. Clinically, EGFR inhibitors are ineffective as single agent compounds in patients with recurrent NB, likely due to this transferred survival dependence to Bcl-2. Likewise, EGFR or ERK inhibitors warrant further testing in combination with Bcl-2 antagonists in vivo as a novel future combination to overcome therapy resistance in the clinic.

Targeting the MDM2–p53 interaction as a therapeutic strategy for the treatment of cancer

Correspondence: Susan K Peirce Department of Pediatrics, Division of Hematology and Oncology emory University School of Medicine, Aflac Cancer Center and Blood Service, 2015 Uppergate Drive, Atlanta, Georgia 30322, USA Tel +1 404 727 4766 Fax +1 404 727-4455 email speirce@emory.edu Abstract: The tumor suppressor p53 functions as an important defense against the development of cancer, and is negatively regulated by interaction with the oncogene and E3 ligase MDM2. In a tightly controlled system of feedback, MDM2 is, in turn, inhibited by the tumor suppressor p14. The inhibition of MDM2-p53 interaction is an appealing therapeutic strategy for the treatment of cancer, and significant advances have been made in the development of small-molecule inhibitors which block this interaction and reactivate wild-type p53. However, the p53 gene is frequently mutated or deleted in cancer, or the wild-type p53 function inhibited by high levels of MDM2. Neuroblastoma (NB) is one such cancer and has presented a major therapeutic challenge in pediatric oncology. Although most NB tumors have wild-type p53, the p14/MDM2/p53 pathway is often altered, leading to resistance to many mainstay chemotherapeutics and a high incidence of relapse. In preclinical studies, the MDM2/p53 interaction inhibitor nutlin-3a has shown effectiveness in the treatment of chemoresistant NB with wild-type, mutant or null-p53 status, indicating that nutlin-3a has potential for the treatment of a broad range of chemoresistant and relapse tumors.

Abstract 5039: EGFR regulates Mcl-1 dependence in chemoresistant and acquired ABT-737-resistant high-risk neuroblastoma (HR NB).

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Patients with HR NB initially respond to treatment yet > 60% die of chemoresistant disease due to inhibited apoptosis. We have shown that HR NBs depend on anti-apoptotic Bcl-2 or Mcl-1 to sequester the pro-death protein Bim for survival. Mice carrying Bcl-2 dependent NB xenografts were cured with the Bcl-2/Bcl-xL antagonist ABT-737 plus cytotoxics, but Mcl-1 dependent NBs were resistant, as ABT-737 does not target Mcl-1. We also showed a Bcl-2 dependent NB cell line (SMS-SAN) selected in vitro to resist high dose ABT-737 (SAN-ABTR) altered Bim binding from Bcl-2 to Mcl-1, despite persistent Bcl-2 expression. SAN-ABTR is also resistant to common cytotoxic drugs compared to SMS-SAN, likely through acquired Mcl-1 dependence. As no current molecules inhibit Mcl-1 directly, we hypothesized that targeting pathways that regulate Mcl-1 expression will restore chemotherapy and Bcl-2 antagonist sensitivity in HR NB. Real time cell sensing (xCELLigence) showed that SAN-ABTR at steady state had a > 2 log increase in proliferation compared to SMS-SAN. Tyrosine kinases (TK) enhance NB proliferation and regulate Mcl-1 in adult cancers, but how they regulate Bcl-2 proteins in NB is unknown. Using phospho-protein microarrays, we found that EGFR and its downstream effectors, AKT, ERK 1/2, and CREB were phosphorylated in SAN-ABTR but not in SMS-SAN. Co-immunoprecipitation (co-IP) showed that inhibition of EGFR by shRNA in SAN-ABTR reverted Bim from Mcl-1 back to Bcl-2 sequestration, despite persistent Mcl-1 expression and restored sensitivity to ABT-737 in vitro. EGFR inhibition also re-sensitized SAN-ABTR to cytotoxic agents. Immunoblots of a panel of 12 NB cell lines characterized for Bcl-2 dependence patterns showed EGFR to be highly expressed in Mcl-1 dependent NBs but not in Bcl-2 dependent NBs. We inhibited EGFR by shRNA in Mcl-1 dependent SKNBE(2) and again Bim moved from Mcl-1 sequestration over to Bcl-2, suggesting a role for EGFR in de novo Mcl-1 dependence. Inhibition of ERK 1/2 (using UO126) resulted in enhanced sensitivity to ABT-737 in Mcl-1 dependent, EGFR expressing NLF cells, suggesting EGFR is regulating Mcl-1:Bim binding through ERK. Work is ongoing to evaluate post-translational modifications causing EGFR mediated Bim:Mcl-1 binding in NB. This data supports a role for EGFR in both pre-existing chemoresistance and emergent targeted therapy resistance in HR NB through modulation of Bcl-2 family interactions. Earlier studies suggest that EGFR inhibitors alone have little utility in NB treatment. Here, we demonstrate that Bcl-2 sequesters Bim upon EGFR inhibitor displacement of Bim from Mcl-1. Therefore, EGFR inhibitors will be more effective if the Bcl-2 survival bias is subverted using Bcl-2 antagonists. The novel targeted combination of EGFR and Bcl-2 blockade has the potential to impact the survival of patients with HR NB as well as other EGFR overexpressing and Mcl-1 dependent cancers. Citation Format: Susan K. Peirce, Srilatha Nalluri, Haneen Abdella, Kelly Goldsmith. EGFR regulates Mcl-1 dependence in chemoresistant and acquired ABT-737-resistant high-risk neuroblastoma (HR NB). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5039. doi:10.1158/1538-7445.AM2013-5039

Abstract 5252: BH3 profiling identifies apoptosis resistance heterogeneity in neuroblastoma and predicts in vivo potency of Bcl2 antagonists

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Patients with high-risk neuroblastoma (NB) frequently succumb to chemoresistant disease that we hypothesize results from deregulation of Bcl2 family proteins. We have shown that NB mitochondria demonstrate specific cytochrome (cyto) c release in response to diverse BH3 peptides. Such “BH3 profiles” identified Bcl2 protein addiction patterns and predicted NB sensitivity to the Bcl2-family antagonists ABT-737 (targeting Bcl2, Bclxl, Bclw) and AT-101 (> Mcl1 avidity) in vitro. We have further assessed whether BH3 profiles are preserved in xenografts and correlate with in vivo therapeutic responses. Heirarchical clustering of BH3 responses from >15 NB profiles identified three groups- “Mcl1 dependence” with a Noxa predominant cyto c release, “Bclxl/Bclw dependence” with a Bik-dominant response, and “BH3 resistance” with markedly blunted cyto c release. BH3 profiles from xenografts tended to co-cluster with their parent monolayer responses. Notably, BH3 resistant NBs are from cell lines derived at relapse after cytotoxic therapy. While most NBs have activated Bim sequestered to pro-survival proteins at steady state, co-IPs show no activated Bim on Mcl1, Bcl2 or Bclxl in relapsed NB cells. This “loss of priming” may explain the extreme therapy resistance seen clinically. In vitro, Bclxl/w dependent SMS-SAN is exquisitely sensitive to combinations of ABT-737 (10 nM) and melphalan, doxorubicin, or etoposide, with cytotoxic IC50s decreased by >1 log. Mcl1 dependent IMR5 cells were also sensitive to cytotoxics plus ABT-737 at 1 uM. BH3 resistant NBs were insensitive to ABT-737 combinations. Real-Time PCR for p53 targets following melphalan treatment show increased Puma, Noxa, and p21 expression in Mcl1 dependent IMR5, but not in BH3 resistant (p53 mutant) SK-N-AS or Bcl-xl/w dependent SMS-SAN, explaining ABT-737/melphalan synergy in IMR5 through possible Noxa/Puma neutralization of Mcl1. Lastly, NB xenografts (XG) from the 3 distinct BH3 profile groups were treated with cyclophosphamide (CTX, 75 mg/kg IPx4), ABT-737 (100 mg/kg IP daily x14), or both drugs. ABT-737 alone partially regressed SMS-SAN XGs, while the ABT-737/CTX combination completely regressed 6/9 SMS-SAN XGs, with cure of 3/9 mice following a single treatment cycle. BH3 profiles from XGs that re-grew showed stable Bclxl/w dependence, and re-treatment led to regression again. Mcl1 dependent XGs were insensitive to ABT-737 combination therapy. The same combination therapy using AT-101 was largely ineffective in all but Mcl1 dependent XGs where marginal benefit was seen. Intact mitochondrial apoptosis is critical to cytotoxic therapy effectiveness. BH3 profiles from NB mitochondria not only reveal mechanisms of relapsed tumor chemoresistance, but accurately identify NBs that benefit from small molecule Bcl2 family inhibition to re-instate chemotherapy potency. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5252.