Kelly C. Goldsmith

Mcl1 downregulation sensitizes neuroblastoma to cytotoxic chemotherapy and small molecule Bcl2-family antagonists

Neuroblastoma (NB) is a common, highly lethal pediatric cancer, with treatment failures largely attributable to the emergence of chemoresistance. The pro-survival Bcl2 homology (BH) proteins critically regulate apoptosis, and may represent important therapeutic targets for restoring drug sensitivity in NB. We used a human NB tumor tissue microarray to survey the expression of pro-survival BH proteins Mcl1 and Bcl2, and correlated expression to clinical prognostic factors and survival. Primary NB tumors heterogeneously expressed Mcl1 or Bcl2, with high expression correlating to high-risk phenotype. Co-expression is infrequent (11%), but correlates to reduced survival. Using RNA interference, we investigated the functional relevance of Mcl1 and Bcl2 in high-risk NB cell lines (SK-N-AS, IMR-5, NLF). Mcl1 knockdown induced apoptosis in all NB cell lines, while Bcl2 knockdown inhibited only NLF, suggesting functional heterogeneity. Finally, we determined the relevance of Mcl1 in resistance to conventional chemotherapy (etoposide, doxorubicin) and small molecule Bcl2-family antagonists (ABT-737 and AT-101). Mcl1 silencing augmented sensitivity to chemotherapeutics 2- to 300-fold, while Bcl2 silencing did not, even in Bcl2-sensitive NLF cells. Resistance to ABT-737, which targets Bcl2/-w/-x, was overcome by Mcl1 knockdown. AT-101, which also neutralizes Mcl1, had single-agent cytotoxicity, further augmented by Mcl1 knockdown. In conclusion, Mcl1 appears a predominant pro-survival protein contributing to chemoresistance in NB, and Mcl1 inactivation may represent a novel therapeutic strategy. Optimization of compounds with higher Mcl1 affinity, or combination with additional Mcl1 antagonists, may enhance the clinical utility of this approach.

BH3 response profiles from neuroblastoma mitochondria predict activity of small molecule Bcl-2 family antagonists.

Bcl-2 family proteins regulate mitochondrial apoptosis downstream of diverse stressors. Cancer cells frequently deregulate Bcl-2 proteins leading to chemoresistance. We have optimized a platform for solid tumors in which Bcl-2 family resistance patterns are inferred. Functional mitochondria were isolated from neuroblastoma (NB) cell lines, exposed to distinct BH3-domain peptides, and assayed for cytochrome c release. Such BH3 profiles revealed three patterns of cytochrome c response. A subset had a dominant NoxaBH3 response implying Mcl1 dependence. These cells were more sensitive to small molecules that antagonize Mcl1 (AT-101) than those that antagonize Bcl-2, Bcl-xL and Bcl-w (ABT-737). A second subset had a dominant BikBH3 response, implying a Bcl-xL/-w dependence, and was exquisitely sensitive to ABT-737 (IC50 <200 nM). Finally, most NB cell lines derived at relapse were relatively resistant to pro-death BH3 peptides and Bcl-2 antagonists. Our findings define heterogeneity for apoptosis resistance in NB, help triage emerging Bcl-2 antagonists for clinical use, and provide a platform for studies to characterize post-therapy resistance mechanisms for NB and other solid tumors.

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.

Multiple components of the spliceosome regulate Mcl1 activity in neuroblastoma

Cancer treatments induce cell stress to trigger apoptosis in tumor cells. Many cancers repress these apoptotic signals through alterations in the Bcl2 proteins that regulate this process. Therapeutics that target these specific survival biases are in development, and drugs that inhibit Bcl2 activities have shown clinical activity for some cancers. Mcl1 is a survival factor for which no effective antagonists have been developed, so it remains a principal mediator of therapy resistance, including to Bcl2 inhibitors. We used a synthetic-lethal screening strategy to identify genes that regulate Mcl1 survival activity using the pediatric tumor neuroblastoma (NB) as a model, as a large subset are functionally verified to be Mcl1 dependent and Bcl2 inhibitor resistant. A targeted siRNA screen identified genes whose knockdown restores sensitivity of Mcl1-dependent NBs to ABT-737, a small molecule inhibitor of Bcl2, BclXL and BclW. Three target genes that shifted the ABT-737 IC50 >1 log were identified and validated: PSMD14, UBL5 and PRPF8. The latter two are members of a recently characterized subcomplex of the spliceosome that along with SART1 is responsible for non-canonical 5′-splice sequence recognition in yeast. We showed that SART1 knockdown similarly sensitized Mcl1-dependent NB to ABT-737 and that triple knockdown of UBL5/PRPF8/SART1 phenocopied direct MCL1 knockdown, whereas having no effect on Bcl2-dependent NBs. Both genetic spliceosome knockdown or treatment with SF3b-interacting spliceosome inhibitors like spliceostatin A led to preferential pro-apoptotic Mcl1-S splicing and reduced translation and abundance of Mcl1 protein. In contrast, BN82865, which inhibits the second transesterification step in terminal spliceosome processing, did not have this effect. These findings demonstrate a prominent role for the spliceosome in mediating Mcl1 activity and suggest that drugs that target either the specific UBL5/PRPF8/SART1 subcomplex or SF3b functions may have a role as cancer therapeutics by attenuating the Mcl1 survival bias present in numerous cancers.

Phase II evaluation of sunitinib in the treatment of recurrent or refractory high-grade glioma or ependymoma in children: a children's Oncology Group Study ACNS1021.

Sunitinib malate is a small multi‐targeted tyrosine kinase inhibitor that inhibits vascular endothelial growth factor receptor (VEGFR), platelet‐derived growth factor receptor (PDGFR) and stem cell factor receptor (KIT), which are highly expressed by some high‐grade brain tumors. We conducted a phase II study to estimate the efficacy and further characterize the pharmacokinetics of sunitinib in pediatric patients with recurrent or refractory high‐grade glioma (Stratum A) or ependymoma (Stratum B). This was a prospective, multicenter Phase II trial conducted through the Childrens Oncology Group (ClinicalTrials.gov Identifier NCT01462695). Sunitinib, 15 mg/m2, was orally administered once daily for 4 weeks every 6 weeks. The safety and tolerability of sunitinib, an estimate of progression‐free survival (PFS), analyses of sunitinib pharmacokinetics (PK) and pharmacodynamics modulation of plasma VEGF and VEGFR2 were also assessed. Thirty eligible patients (17 patients on Stratum A, 13 patients on Stratum B) were enrolled and 29 patients were evaluable for response. Sunitinib was reasonably well tolerated in children with recurrent ependymoma or high‐grade glioma. Most adverse events were of mild‐to‐moderate severity and manageable with supportive treatment. While there was a statistically significant modulation of plasma VEGFR2 with sunitinib exposure, there were no sustained tumor responses. Both strata were closed at time of planned interim analysis as there was not sufficient efficacy associated with sunitinib in children with recurrent brain tumors. Sunitinib was well tolerated in children and young adults with recurrent high‐grade glioma or ependymoma but had no single agent objective antitumor activity in these patients.

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.

Select Bcl-2 antagonism restores chemotherapy sensitivity in high-risk neuroblastoma

BackgroundPediatric patients with high-risk neuroblastoma (HR NB) often fail to respond to upfront intensive multimodal therapy. Tumor-acquired suppression of apoptosis contributes to therapy resistance. Many HR NB tumors depend on the anti-apoptotic protein Bcl-2 for survival, through Bcl-2 sequestration and inhibition of the pro-apoptotic protein, Bim. Bcl-2 dependent xenografts derived from aggressive human NB tumors are cured with a combination of cyclophosphamide and ABT-737, a Bcl-2/Bcl-XL/Bcl-w small molecule antagonist. The oral analogue to ABT-737, Navitoclax (ABT-263), clinically causes an immediate drop in peripheral platelet counts as mature platelets depend on Bcl-xL for survival. This led to the creation of a Bcl-2 selective inhibitor, ABT-199 (Venetoclax). A Phase I trial of ABT-199 in CLL showed remarkable antitumor activity and stable patient platelet counts. Given Bcl-XL does not play a role in HR NB survival, we hypothesized that ABT-199 would be equally potent against HR NB.MethodsCytotoxicity and apoptosis were measured in human derived NB cell lines exposed to ABT-199 combinations. Co-Immunoprecipitation evaluated Bim displacement from Bcl-2, following ABT-199. Murine xenografts of NB cell lines were grown and then exposed to a 14-day course of ABT-199 alone and with cyclophosphamide.ResultsBcl-2 dependent NB cell lines are exquisitely sensitive to ABT-199 (IC50 1.5–5 nM) in vitro, where Mcl-1 dependent NBs are completely resistant. Treatment with ABT-199 displaces Bim from Bcl-2 in NB to activate caspase 3, confirming the restoration of mitochondrial apoptosis. Murine xenografts of Mcl-1 and Bcl-2 dependent NBs were treated with a two-week course of ABT-199, cyclophosphamide, or ABT-199/cyclophosphamide combination. Mcl-1 dependent tumors did not respond to ABT-199 alone and showed no significant difference in time to tumor progression between chemotherapy alone or ABT-199/cyclophosphamide combination. In contrast, Bcl-2 dependent xenografts responded to ABT-199 alone and had sustained complete remission (CR) to the ABT-199/cyclophosphamide combination, with one recurrent tumor maintaining Bcl-2 dependence and obtaining a second CR after a second course of therapy.ConclusionHR NB patients are often thrombocytopenic at relapse, raising concerns for therapies like ABT-263 despite its HR NB tumor targeting potential. Our data confirms that Bcl-2 selective inhibitors like ABT-199 are equally potent in HR NB in vitro and in vivo and given their lack of platelet toxicity, should be translated into the clinic for HR NB.

Concurrent myeloid sarcoma, atypical teratoid/rhabdoid tumor, and hypereosinophilia in an infant with a germline SMARCB1 mutation

We report a 1‐year‐old female child presenting with hypereosinophilia who was found to have concurrent myeloid sarcoma and a central nervous system (CNS) atypical teratoid/rhabdoid tumor (AT/RT). She was later found to have a germline mutation in SMARCB1. Concurrent hematologic malignancy and CNS AT/RT have not previously been described in the context of a SMARCB1 loss‐of‐function germline mutation.

Hyperleukocytosis in infant acute leukemia: a role for manual exchange transfusion for leukoreduction: INFANT LEUKEMIA MANUAL EXCHANGE TRANSFUSION

Hyperleukocytosis is a serious, life‐threatening complication of pediatric acute leukemia that can cause neurologic injury, pulmonary leukostasis, metabolic derangements, and coagulopathy. Acute leukemia has the highest risk of mortality and morbidity at presentation when associated with hyperleukocytosis. Infant leukemia presents unique challenges and treatment considerations due to the disease itself and size and overall health of the patient. While medical management of hyperleukocytosis in older patients with acute leukemia has been described, including cytoreductive procedures with automated leukapheresis (AL) or manual whole blood (WB) exchange transfusion, very little data exist for standardized management of hyperleukocytosis in infant leukemia patients.

Accelerating drug development in pediatric cancer: a novel Phase I study design of venetoclax in relapsed/refractory malignancies

Venetoclax is a highly selective, potent BCL-2 inhibitor that is approved for some patients previously treated for chronic lymphocytic leukemia, and has shown promising activity in adult studies across several hematologic malignancies. Preclinical studies have demonstrated venetoclax activity in pediatric patient-derived xenograft models and cell lines; however, clinical studies in pediatric patients have yet to be conducted. The prognosis is poor for children with most relapsed/refractory malignancies, and limited treatment options result in unmet clinical need. Herein, we describe the rationale and design of the first study of venetoclax in pediatric patients with relapsed/refractory malignancies: a Phase I trial investigating the safety and pharmacokinetics of venetoclax monotherapy followed by the addition of chemotherapy (Trial registration: EudraCT 2017-000439-14; NCT03236857).