Phase 1 study of sirolimus in combination with oral cyclophosphamide and topotecan in children and young adults with relapsed and refractory solid tumors.

PurposeTo determine the maximum tolerated dose (MTD), toxicities, and pharmacodynamics effects of sirolimus combined with oral metronomic topotecan and cyclophosphamide in a pediatric population.Materials and methodsPatients who were 1 to 30 years of age with relapsed/refractory solid tumors (including CNS) were eligible. Patients received daily oral sirolimus and cyclophosphamide (25-50 mg/m2/dose) on days 1-21 and oral topotecan (0.8 mg/m2/dose) on days 1-14 in 28-day cycles. Sirolimus steady-state plasma trough concentrations of 3-7.9 ng/mL and 8-12.0 ng/mL were evaluated, with dose escalation based on a 3+3 phase 1 design. Biomarkers of angiogenesis were also evaluated.ResultsTwenty-one patients were treated (median age 18 years; range 9-30). Dose-limiting toxicities included myelosuppression, ALT elevation, stomatitis, and hypertriglyceridemia. The MTD was sirolimus with trough goal of 8-12.0 ng/mL; cyclophosphamide 25 mg/m2/dose; and topotecan 0.8 mg/m2/dose. No objective responses were observed. Four patients had prolonged stable disease > 4 cycles (range 4-12). Correlative biomarker analyses demonstrated reductions in thrombospondin-1 (p=0.043) and soluble vascular endothelial growth factor receptor-2 plasma concentrations at 21 days compared to baseline.ConclusionsThe combination of oral sirolimus, topotecan, and cyclophosphamide was well tolerated and biomarker studies demonstrated modulation of angiogenic pathways with this regimen

ALK upregulates POSTN and WNT signaling to drive neuroblastoma

Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling

ALK upregulates POSTN and WNT signaling to drive neuroblastoma

Summary: Neuroblastoma is the most common extracranial solid tumor of childhood. While MYCN and mutant anaplastic lymphoma kinase (ALKF1174L) cooperate in tumorigenesis, how ALK contributes to tumor formation remains unclear. Here, we used a human stem cell-based model of neuroblastoma. Mis-expression of ALKF1174L and MYCN resulted in shorter latency compared to MYCN alone. MYCN tumors resembled adrenergic, while ALK/MYCN tumors resembled mesenchymal, neuroblastoma. Transcriptomic analysis revealed enrichment in focal adhesion signaling, particularly the extracellular matrix genes POSTN and FN1 in ALK/MYCN tumors. Patients with ALK-mutant tumors similarly demonstrated elevated levels of POSTN and FN1. Knockdown of POSTN, but not FN1, delayed adhesion and suppressed proliferation of ALK/MYCN tumors. Furthermore, loss of POSTN reduced ALK-dependent activation of WNT signaling. Reciprocally, inhibition of the WNT pathway reduced expression of POSTN and growth of ALK/MYCN tumor cells. Thus, ALK drives neuroblastoma in part through a feedforward loop between POSTN and WNT signaling

Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma

Background Glioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed. Methods We used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations. Results ICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation. Conclusions Our data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo

Resistance to Epigenetic-Targeted Therapy Engenders Tumor Cell Vulnerabilities Associated with Enhancer Remodeling

Drug resistance represents a major challenge to achieving durable responses to cancer therapeutics. Resistance mechanisms to epigenetically targeted drugs remain largely unexplored. We used bromodomain and extra-terminal domain (BET) inhibition in neuroblastoma as a prototype to model resistance to chromatin modulatory therapeutics. Genome-scale, pooled lentiviral open reading frame (ORF) and CRISPR knockout rescue screens nominated the phosphatidylinositol 3-kinase (PI3K) pathway as promoting resistance to BET inhibition. Transcriptomic and chromatin profiling of resistant cells revealed that global enhancer remodeling is associated with upregulation of receptor tyrosine kinases (RTKs), activation of PI3K signaling, and vulnerability to RTK/PI3K inhibition. Large-scale combinatorial screening with BET inhibitors identified PI3K inhibitors among the most synergistic upfront combinations. These studies provide a roadmap to elucidate resistance to epigenetic-targeted therapeutics and inform efficacious combination therapies. Using functional screens, profiling of drug-resistant cells, and drug combination screens in neuroblastoma, Iniguez et al. show that PI3K pathway activation via enhancer remodeling and transcriptional reprogramming confers resistance to BET inhibitors (BETi) and that PI3K inhibitors synergize with BETi

CRISPR-Cas9 screen reveals a MYCN-amplified neuroblastoma dependency on EZH2

Pharmacologically difficult targets, such as MYC transcription factors, represent a major challenge in cancer therapy. For the childhood cancer neuroblastoma, amplification of the oncogene MYCN is associated with high-risk disease and poor prognosis. Here, we deployed genome-scale CRISPR-Cas9 screening of MYCN-amplified neuroblastoma and found a preferential dependency on genes encoding the polycomb repressive complex 2 (PRC2) components EZH2, EED, and SUZ12. Genetic and pharmacological suppression of EZH2 inhibited neuroblastoma growth in vitro and in vivo. Moreover, compared with neuroblastomas without MYCN amplification, MYCN-amplified neuroblastomas expressed higher levels of EZH2. ChIP analysis showed that MYCN binds at the EZH2 promoter, thereby directly driving expression. Transcriptomic and epigenetic analysis, as well as genetic rescue experiments, revealed that EZH2 represses neuronal differentiation in neuroblastoma in a PRC2-dependent manner. Moreover, MYCN-amplified and high-risk primary tumors from patients with neuroblastoma exhibited strong repression of EZH2-regulated genes. Additionally, overexpression of IGFBP3, a direct EZH2 target, suppressed neuroblastoma growth in vitro and in vivo. We further observed strong synergy between histone deacetylase inhibitors and EZH2 inhibitors. Together, these observations demonstrate that MYCN upregulates EZH2, leading to inactivation of a tumor suppressor program in neuroblastoma, and support testing EZH2 inhibitors in patients with MYCN-amplified neuroblastoma