Joshua B. Rubin

A small-molecule antagonist of CXCR4 inhibits intracranial growth of primary brain tumors

The vast majority of brain tumors in adults exhibit glial characteristics. Brain tumors in children are diverse: Many have neuronal characteristics, whereas others have glial features. Here we show that activation of the Gi protein-coupled receptor CXCR4 is critical for the growth of both malignant neuronal and glial tumors. Systemic administration of CXCR4 antagonist AMD 3100 inhibits growth of intracranial glioblastoma and medulloblastoma xenografts by increasing apoptosis and decreasing the proliferation of tumor cells. This reflects the ability of AMD 3100 to reduce the activation of extracellular signal-regulated kinases 1 and 2 and Akt, all of which are pathways downstream of CXCR4 that promote survival, proliferation, and migration. These studies (i) demonstrate that CXCR4 is critical to the progression of diverse brain malignances and (ii) provide a scientific rationale for clinical evaluation of AMD 3100 in treating both adults and children with malignant brain tumors.

Targeting the leukemia microenvironment by CXCR4 inhibition overcomes resistance to kinase inhibitors and chemotherapy in AML

SDF-1alpha/CXCR4 signaling plays a key role in leukemia/bone marrow microenvironment interactions. We previously reported that bone marrow-derived stromal cells inhibit chemotherapy-induced apoptosis in acute myeloid leukemia (AML). Here we demonstrate that the CXCR4 inhibitor AMD3465 antagonized stromal-derived factor 1alpha (SDF-1alpha)-induced and stroma-induced chemotaxis and inhibited SDF-1alpha-induced activation of prosurvival signaling pathways in leukemic cells. Further, CXCR4 inhibition partially abrogated the protective effects of stromal cells on chemotherapy-induced apoptosis in AML cells. Fetal liver tyrosine kinase-3 (FLT3) gene mutations activate CXCR4 signaling, and coculture with stromal cells significantly diminished antileukemia effects of FLT3 inhibitors in cells with mutated FLT3. Notably, CXCR4 inhibition increased the sensitivity of FLT3-mutated leukemic cells to the apoptogenic effects of the FLT3 inhibitor sorafenib. In vivo studies demonstrated that AMD3465, alone or in combination with granulocyte colony-stimulating factor, induced mobilization of AML cells and progenitor cells into circulation and enhanced antileukemic effects of chemotherapy and sorafenib, resulting in markedly reduced leukemia burden and prolonged survival of the animals. These findings indicate that SDF-1alpha/CXCR4 interactions contribute to the resistance of leukemic cells to signal transduction inhibitor- and chemotherapy-induced apoptosis in systems mimicking the physiologic microenvironment. Disruption of these interactions with CXCR4 inhibitors represents a novel strategy of sensitizing leukemic cells by targeting their protective bone marrow microenvironment.

Intensive Multimodality Treatment for Children With Newly Diagnosed CNS Atypical Teratoid Rhabdoid Tumor

PURPOSE Atypical teratoid rhabdoid tumor (ATRT) of the CNS is a highly malignant neoplasm primarily affecting young children, with a historic median survival ranging from 6 to 11 months. Based on a previous pilot series, a prospective multi-institutional trial was conducted for patients with newly diagnosed CNS ATRT. PATIENTS AND METHODS Treatment was divided into five phases: preirradiation, chemoradiation, consolidation, maintenance, and continuation therapy. Intrathecal chemotherapy was administered, alternating intralumbar and intraventricular routes. Radiation therapy (RT) was prescribed, either focal (54 Gy) or craniospinal (36 Gy, plus primary boost), depending on age and extent of disease at diagnosis. RESULTS Between 2004 and 2006, 25 patients were enrolled; 20 were eligible for evaluation. Median age at diagnosis was 26 months (range, 2.4 months to 19.5 years). Gross total resection of the primary tumor was achieved in 11 patients. Fourteen patients had M0 disease at diagnosis, one patient had M2 disease, and five patients had M3 disease. Fifteen patients received radiation therapy: 11 focal and four craniospinal. Significant toxicities, in addition to the expected, included radiation recall (n = 2) and transverse myelitis (n = 1). There was one toxic death. Of the 12 patients who were assessable for chemotherapeutic response (pre-RT), the objective response rate was 58%. The objective response rate observed after RT was 38%. The 2-year progression-free and overall survival rates are 53% +/- 13% and 70% +/- 10%, respectively. Median overall survival has not yet been reached. CONCLUSION This intensive multimodality regimen has resulted in a significant improvement in time to progression and overall survival for patients with this previously poor-prognosis tumor.

Recurrent somatic mutations in ACVR1 in pediatric midline high-grade astrocytoma

Pediatric midline high-grade astrocytomas (mHGAs) are incurable with few treatment targets identified. Most tumors harbor mutations encoding p.Lys27Met in histone H3 variants. In 40 treatment-naive mHGAs, 39 analyzed by whole-exome sequencing, we find additional somatic mutations specific to tumor location. Gain-of-function mutations in ACVR1 occur in tumors of the pons in conjunction with histone H3.1 p.Lys27Met substitution, whereas FGFR1 mutations or fusions occur in thalamic tumors associated with histone H3.3 p.Lys27Met substitution. Hyperactivation of the bone morphogenetic protein (BMP)-ACVR1 developmental pathway in mHGAs harboring ACVR1 mutations led to increased levels of phosphorylated SMAD1, SMAD5 and SMAD8 and upregulation of BMP downstream early-response genes in tumor cells. Global DNA methylation profiles were significantly associated with the p.Lys27Met alteration, regardless of the mutant histone H3 variant and irrespective of tumor location, supporting the role of this substitution in driving the epigenetic phenotype. This work considerably expands the number of potential treatment targets and further justifies pretreatment biopsy in pediatric mHGA as a means to orient therapeutic efforts in this disease.

A feasibility trial of antiangiogenic (metronomic) chemotherapy in pediatric patients with recurrent or progressive cancer.

Standard chemotherapeutic drugs, when modified by the frequency and dose of administration, can target angiogenesis. This approach is referred to as antiangiogenic chemotherapy, low-dose chemotherapy, or metronomic chemotherapy. This study evaluated the feasibility of 6 months of metronomic chemotherapy, its toxicity and tolerability, surrogate markers of activity, and preliminary evidence of activity in children with recurrent or progressive cancer. Twenty consecutive children were enrolled and received continuous oral thalidomide and celecoxib with alternating oral etoposide and cyclophosphamide every 21 days for a planned duration of 6 months using antiangiogenic doses of all four drugs. Surrogate markers including bFGF, VEGF, endostatin, and thrombospondin were also evaluated. Therapy was well tolerated in this heavily pretreated population. Toxicities (predominantly reversible bone marrow suppression) responded to dose modifications. Sixty percent of the patients received less than the prescribed 6 months of therapy due to toxicity (one case of deep vein thrombosis), personal choice (1 patient), or disease progression (10 patients). Forty percent of the patients completed the 6 months of therapy, resulting in prolonged or persistent disease-free status. One quarter of all patients continue to be progression free more than 123 weeks from starting therapy. Sixteen percent of patients showed a radiographic partial response. Only elevated thrombospondin-1 levels appeared to correlate with prolonged response. This oral antiangiogenic chemotherapy regimen was well tolerated in this heavily pretreated pediatric population, which showed prolonged or persistent disease-free status, supporting the continued study of antiangiogenic/metronomic chemotherapy in human clinical trials.

Subgroup-Specific Prognostic Implications of TP53 Mutation in Medulloblastoma

PURPOSE Reports detailing the prognostic impact of TP53 mutations in medulloblastoma offer conflicting conclusions. We resolve this issue through the inclusion of molecular subgroup profiles. PATIENTS AND METHODS We determined subgroup affiliation, TP53 mutation status, and clinical outcome in a discovery cohort of 397 medulloblastomas. We subsequently validated our results on an independent cohort of 156 medulloblastomas. RESULTS TP53 mutations are enriched in wingless (WNT; 16%) and sonic hedgehog (SHH; 21%) medulloblastomas and are virtually absent in subgroups 3 and 4 tumors (P < .001). Patients with SHH/TP53 mutant tumors are almost exclusively between ages 5 and 18 years, dramatically different from the general SHH distribution (P < .001). Children with SHH/TP53 mutant tumors harbor 56% germline TP53 mutations, which are not observed in children with WNT/TP53 mutant tumors. Five-year overall survival (OS; ± SE) was 41% ± 9% and 81% ± 5% for patients with SHH medulloblastomas with and without TP53 mutations, respectively (P < .001). Furthermore, TP53 mutations accounted for 72% of deaths in children older than 5 years with SHH medulloblastomas. In contrast, 5-year OS rates were 90% ± 9% and 97% ± 3% for patients with WNT tumors with and without TP53 mutations (P = .21). Multivariate analysis revealed that TP53 status was the most important risk factor for SHH medulloblastoma. Survival rates in the validation cohort mimicked the discovery results, revealing that poor survival of TP53 mutations is restricted to patients with SHH medulloblastomas (P = .012) and not WNT tumors. CONCLUSION Subgroup-specific analysis reconciles prior conflicting publications and confirms that TP53 mutations are enriched among SHH medulloblastomas, in which they portend poor outcome and account for a large proportion of treatment failures in these patients.

Pathological Expression of CXCL12 at the Blood-Brain Barrier Correlates with Severity of Multiple Sclerosis

Dysregulation of blood-brain barrier (BBB) function and transendothelial migration of leukocytes are essential components of the development and propagation of active lesions in multiple sclerosis (MS). Animal studies indicate that polarized expression of the chemokine CXCL12 at the BBB prevents leukocyte extravasation into the central nervous system (CNS) and that disruption of CXCL12 polarity promotes entry of autoreactive leukocytes and inflammation. In the present study, we examined expression of CXCL12 and its receptor, CXCR4, within CNS tissues from MS and non-MS patients. Immunohistochemical analysis of CXCL12 expression at the BBB revealed basolateral localization in tissues derived from non-MS patients and at uninvolved sites in tissues from MS patients. In contrast, within active MS lesions, CXCL12 expression was redistributed toward vessel lumena and was associated with CXCR4 activation in infiltrating leukocytes, as revealed by phospho-CXCR4-specific antibodies. Quantitative assessment of CXCL12 expression by the CNS microvasculature established a positive correlation between CXCL12 redistribution, leukocyte infiltration, and severity of histological disease. These results suggest that CXCL12 normally functions to localize infiltrating leukocytes to perivascular spaces, preventing CNS parenchymal infiltration. In the patient cohort studied, altered patterns of CXCL12 expression at the BBB were specifically associated with MS, possibly facilitating trafficking of CXCR4-expressing mononuclear cells into and out of the perivascular space and leading to progression of disease.

Cytogenetic Prognostication Within Medulloblastoma Subgroups

PURPOSE Medulloblastoma comprises four distinct molecular subgroups: WNT, SHH, Group 3, and Group 4. Current medulloblastoma protocols stratify patients based on clinical features: patient age, metastatic stage, extent of resection, and histologic variant. Stark prognostic and genetic differences among the four subgroups suggest that subgroup-specific molecular biomarkers could improve patient prognostication. PATIENTS AND METHODS Molecular biomarkers were identified from a discovery set of 673 medulloblastomas from 43 cities around the world. Combined risk stratification models were designed based on clinical and cytogenetic biomarkers identified by multivariable Cox proportional hazards analyses. Identified biomarkers were tested using fluorescent in situ hybridization (FISH) on a nonoverlapping medulloblastoma tissue microarray (n = 453), with subsequent validation of the risk stratification models. RESULTS Subgroup information improves the predictive accuracy of a multivariable survival model compared with clinical biomarkers alone. Most previously published cytogenetic biomarkers are only prognostic within a single medulloblastoma subgroup. Profiling six FISH biomarkers (GLI2, MYC, chromosome 11 [chr11], chr14, 17p, and 17q) on formalin-fixed paraffin-embedded tissues, we can reliably and reproducibly identify very low-risk and very high-risk patients within SHH, Group 3, and Group 4 medulloblastomas. CONCLUSION Combining subgroup and cytogenetic biomarkers with established clinical biomarkers substantially improves patient prognostication, even in the context of heterogeneous clinical therapies. The prognostic significance of most molecular biomarkers is restricted to a specific subgroup. We have identified a small panel of cytogenetic biomarkers that reliably identifies very high-risk and very low-risk groups of patients, making it an excellent tool for selecting patients for therapy intensification and therapy de-escalation in future clinical trials.

Blocking CXCR4-Mediated Cyclic AMP Suppression Inhibits Brain Tumor Growth In vivo

The chemokine CXCL12 and its cognate receptor CXCR4 regulate malignant brain tumor growth and are potential chemotherapeutic targets. However, the molecular basis for CXCL12-induced tumor growth remains unclear, and the optimal approach to inhibiting CXCR4 function in cancer is unknown. To develop such a therapeutic approach, we investigated the signaling pathways critical for CXCL12 function in normal and malignant cells. We discovered that CXCL12-dependent tumor growth is dependent upon sustained inhibition of cyclic AMP (cAMP) production, and that the antitumor activity of the specific CXCR4 antagonist AMD 3465 is associated with blocking cAMP suppression. Consistent with these findings, we show that pharmacologic elevation of cAMP with the phosphodiesterase inhibitor Rolipram suppresses tumor cell growth in vitro and, upon oral administration, inhibits intracranial growth in xenograft models of malignant brain tumors with comparable efficacy to AMD 3465. These data indicate that the clinical evaluation of phosphodiesterase inhibitors in the treatment of patients with brain tumors is warranted.

Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32

Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.