PurposeThe prognosis for patients diagnosed with glioblastoma multiforme (GBM) remains dismal, with current treatment prolonging survival only modestly. As such, there remains a strong need for novel therapeutic strategies. The janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 pathway regulates many cellular processes in GBM, including survival, proliferation, invasion, anti-apoptosis, and immune evasion. Here, we evaluated the preclinical efficacy of pacritinib, a novel compound targeting JAK2, using a collection of diverse patient-derived brain tumor initiating cells (BTICs).Experimental designThe effects of pacritinib on BTIC viability and sphere forming capacity were evaluated in vitro using the alamarBlue and neurosphere assays, respectively. On-target inhibition of JAK2/STAT3 signaling was investigated using western blotting. The efficacy of pacritinib was tested in vivo in pharmacokinetic analyses, liver microsome analyses, and Kaplan-Meier survival studies.ResultsIn vitro, pacritinib decreased BTIC viability and sphere forming potential at low micromolar doses and demonstrated on-target inhibition of STAT3 signaling. Additionally, pacritinib was found to improve the response to temozolomide (TMZ) in TMZ-resistant BTICs. In vivo, systemic treatment with pacritinib demonstrated blood-brain barrier penetration and led to improved overall median survival in combination with TMZ, in mice orthotopically xenografted with an aggressive recurrent GBM BTIC culture.ConclusionThis preclinical study demonstrates the efficacy of pacritinib and supports the feasibility of testing pacritinib for the treatment of GBM, in combination with the standard of care TMZ.

In glioblastoma (GBM), brain tumor stem cells (BTSCs) encompass heterogenous populations of multipotent, self-renewing, and tumorigenic cells, which have been proposed to be at the root of therapeutic resistance and recurrence. While the functional significance of BTSC heterogeneity remains to be fully determined, we previously distinguished relatively quiescent stem-like precursor state from the more aggressive progenitor-like precursor state. In the present study, we hypothesized that progenitor-like BTSCs arise from stem-like precursors through a mesenchymal transition and drive post-treatment recurrence. We first demonstrate that progenitor-like BTSCs display a more mesenchymal transcriptomic profile. Moreover, we show that both mesenchymal GBMs and progenitor-like BTSCs are characterized by over-activated STAT3/EMT pathways and that SLUG is the primary epithelial to mesenchymal transition (EMT) transcription factor directly regulated by STAT3 in BTSCs. SLUG overexpression in BTSCs enhances invasiveness, promotes inflammation, and shortens survival. Importantly, SLUG overexpression in a quiescent stem-like BTSC line enhances tumorigenesis. Finally, we report that recurrence is associated with SLUG-induced transcriptional changes in both BTSCs and GBM patient samples. Collectively, our findings show that a STAT3-driven precursor state transition, mediated by SLUG, may prime BTSCs to initiate more aggressive mesenchymal recurrence. Targeting the STAT3/SLUG pathway may maintain BTSCs in a quiescent stem-like precursor state, delaying recurrence and improving survival in GBM.

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here we report that, across a panel of 19 glioblastoma BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. Statement of Significance: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death. Research.

Background The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including GBM. In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification status of MDM2, in vitro and in orthotopic xenograft models. Methods In vitro growth inhibition and on-target efficacy of BI-907828 was assessed by cell viability, co-immunoprecipitation assays and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry and in intracranial xenograft models. Results BI-907828 decreases viability and induces cell death at picomolar concentrations, in both MDM2 amplified and normal copy number, TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. PK-PD studies in orthotopic tumor-bearing SCID mice demonstrated that a single 50mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. Conclusions BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.