On-target JAK2/STAT3 inhibition slows disease progression in orthotopic xenografts of human glioblastoma brain tumor stem cells

Glioblastoma multiforme (GBM) is a devastating disease that is notoriously resistant to current therapies, leading to dismal patient outcomes and a median survival of just 14.6 months.1 As such, translation of our rapidly expanding knowledge of the molecular biology of GBM together with the growing repertoire of molecular-based chemotherapeutics now available has the potential to dramatically improve patient outcomes. Signal transducer and activator of transcription (STAT)3 signaling has recently come to the forefront of the glioma literature as a common pathway downstream of paracrine leukemia inhibitory factor, interleukin-6, or erythropoietin signaling in GBM.2–4 Recent transcriptional profiling and signaling network analyses have also identified STAT3 and CCAAT (cytidine-cytidine-adenosine-adenosine-thymidine)-enhancer-binding proteins β as 2 key master transcription factors underlying the mesenchymal subtype of GBM.5 Consequently, STAT3 has become a therapeutic target of significant interest in glioma, and a number of small-molecule inhibitors of the STAT3' upstream regulator Janus kinase (JAK)2 are currently in early preclinical investigation in GBM. Inhibition of JAK2 with AG490 or its more potent second-generation analog WP1066 has demonstrated efficacy in reducing survival and proliferation of U87 and U251 glioma cells in vitro and decreasing tumorigenicity of U87 in subcutaneous xenografts.6,7 JAK2/STAT3 blockade has also demonstrated promise as an adjuvant for other therapeutics. Concurrent inhibition of JAK2/STAT3 with cucurbitacin-I (JSI-124) and blockade of epidermal growth factor receptor (EGFR) with Iressa has been recently reported to potently induce apoptosis in GBM cell lines and sensitize them to temozolomide.8 Emerging evidence suggests that high levels of STAT3 activation are also present within brain tumor stem cells (BTSCs), and STAT3 inhibition has been reported to decrease the self-renewal survival and resistance to temozolomide of glioma stemlike cells in vitro.2–4,9,10 Thus, there is strong evidence in the glioma literature for the crucial role of STAT3 in human GBM, and JAK2-targeted therapeutics have yielded promising results against GBM cell lines in vitro. However, the true translational potential of JAK2/STAT3 therapeutics for GBM remained an unanswered question, as these drugs had yet to be thoroughly investigated in a model that accounted for the molecular heterogeneity of human GBM and assessed on-target efficacy in intracranial humanlike GBM tumors in vivo. BTSCs have been postulated to be at the root of the disease initiation, recurrence, and therapeutic resistance that is characteristic of GBM. BTSCs isolated from human GBM tumors capture the molecular heterogeneity and mutation spectrum that are characteristic of the disease.11,12 BTSCs also initiate orthotopic xenograft tumors in nonobese diabetic/severe combined immunodeficient (NOD SCID) mice that emulate the phenotypic and molecular characteristics of their parent human tumors, hence making them the most relevant model currently available for preclinical evaluation of novel GBM therapeutics. Our group has successfully established a large collection of BTSC lines from glioma patients that display the molecular heterogeneity of the disease,13 including rare lines endogenously expressing isocitrate dehydrogenase (IDH1)R132H, differential MGMT promoter methylation status, and mutations in common targets such as PTEN, TP53, and EGFR variant III (unpublished), amongst others.14–16 Here, we use a large panel of patient-derived GBM BTSCs to investigate whether JAK2/STAT3 is (i) a clinically relevant signaling pathway whose activity is crucial in molecularly diverse BTSCs and (ii) amenable to therapeutic targeting in vivo in orthotopic humanlike GBM xenografts. We demonstrate that JAK2/STAT3 signaling is a key driver for proliferation in GBM BTSCs and that on-target inhibition of this pathway results in decreased BTSC survival in vitro and slows disease progression in vivo.