Background Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves maximal surgical resection of the tumor followed by radiation and chemotherapy (temozolomide [TMZ]). The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status in the tumor. Methods A drug screening aimed at evaluating the potential recycling and repurposing of known drugs was conducted in TMZ-resistant GBM cell lines and primary cultures of newly diagnosed GBM with different MGMT promoter methylation status, phenotypic/genotypic background and subtype, and validated with sphere formation, cell migration assays, and quantitative invasive orthotopic in vivo models. Results We identified hydroxyurea (HU) to synergize with TMZ in GBM cells in culture and in vivo, irrespective of MGMT promoter methylation status, subtype, and/or stemness. HU acts specifically on the S-phase of the cell cycle by inhibiting the M2 unit of enzyme ribonucleotide reductase. Knockdown of this enzyme using RNA interference and other known chemical inhibitors exerted a similar effect to HU in combination with TMZ both in culture and in vivo. Conclusions We demonstrate preclinical efficacy of repurposing hydroxyurea in combination with TMZ for adjuvant GBM therapy. This combination benefit is of direct clinical interest given the extensive use of TMZ and the associated problems with TMZ-related resistance and treatment failure.

Abstract Infant high-grade gliomas appear clinically distinct from their counterparts in older children, indicating that histopathologic grading may not accurately reflect the biology of these tumors. We have collected 241 cases under 4 years of age, and carried out histologic review, methylation profiling, and custom panel, genome, or exome sequencing. After excluding tumors representing other established entities or subgroups, we identified 130 cases to be part of an “intrinsic” spectrum of disease specific to the infant population. These included those with targetable MAPK alterations, and a large proportion of remaining cases harboring gene fusions targeting ALK (n = 31), NTRK1/2/3 (n = 21), ROS1 (n = 9), and MET (n = 4) as their driving alterations, with evidence of efficacy of targeted agents in the clinic. These data strongly support the concept that infant gliomas require a change in diagnostic practice and management. Significance: Infant high-grade gliomas in the cerebral hemispheres comprise novel subgroups, with a prevalence of ALK, NTRK1/2/3, ROS1, or MET gene fusions. Kinase fusion–positive tumors have better outcome and respond to targeted therapy clinically. Other subgroups have poor outcome, with fusion-negative cases possibly representing an epigenetically driven pluripotent stem cell phenotype. See related video: https://vimeo.com/438254885 See related commentary by Szulzewsky and Cimino, p. 904. This article is highlighted in the In This Issue feature, p. 890

Infant gliomas have been considered as early-onset examples of tumour types found throughout paediatric populations. Interestingly, infant high-grade gliomas (HGG) have a better overall survival compared to older children, indicating that grading may not accurately reflect the biology of these tumours. We have to-date collected 181 cases of diffuse glioma (WHO II-IV) occurring in children aged <4 years for histological review, methylation profiling, gene fusion panel and whole genome/exome sequencing. Of 147 samples profiled using the Heidelberg methylation classifier, 20 were reassigned as established brain tumour entities (e.g. pilocytic astrocytoma, HGNET-BCOR), whilst a further 23 fell into recognised HGG subgroups. The remaining 104 cases formed four distinct subgroups by consensus clustering. Two groups were mostly classified as ‘infantile hemispheric gliomas’ (IHG), one of these showing enrichment for ALK/NTRK fusion genes, the other a male predominance, and both with a median age ≤1 year. IHG had a highly cellular, uniform architecture with gemistocytic-like or spindle cell morphology. A third group did not fit with established methylation subgroups but were largely female, ≤6 months, and harboured ALK/NTRK fusions. The fourth group were older, comprising desmoplastic infantile gliomas and poorly-scoring cases of other low-grade tumours. We established three novel patient-derived cultures of IHG, with TPM3:NTRK1, KCTD16:NTRK2 and ETV6:NTRK3 fusions. Drug screening with multiple NTRK inhibitors revealed marked sensitivity in vitro, making such patients excellent candidates for clinical trials of these compounds. The clinical, histological and molecular features of infant gliomas suggest the presence of novel tumour subgroups, which may have targetable driving alterations.

Diffuse intrinsic pontine gliomas (DIPGs) are highly aggressive pediatric brain tumors that are characterized by a recurrent mutation (K27M) within the histone H3 encoding genes H3F3A or HIST1H3A/B/C. These mutations have been shown to induce a global reduction in the repressive histone modification H3K27me3. Together with global changes in DNA methylation patterns this results in a broad diversity of downstream effects which hampers the identification of single therapeutic targets based on a molecular rationale. Therefore, our aim is to identify critical nodes of tumor development and maintenance using a DECIPHER pooled shRNA screening approach in combination with next-generation sequencing. The shRNA library targets more than 5,000 genes, with multiple shRNAs per target as well as internal controls. Currently, four patient-derived H3.3K27M mutated DIPG vs. two H3.3WT control high-grade glioma cell lines are being screened for shRNAs inducing cell death. Preliminary hits unexpectedly included the H3.3-specific histone chaperone complex components ATRX (alpha thalassemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein) - targets which are sometimes mutated in DIPG. This suggests a more complex role of H3.3-dependent heterochromatin formation in DIPGs than is currently appreciated. These and other promising hits will be validated in an expanded cell line panel using a custom CRISPR/Cas9 library, with top candidates further taken forward to in vivo pre-clinical testing for identification of drug targets.

Treatment of medulloblastoma in children fails in approximately 30% of patients, and is often accompanied by severe late sequelae. Therefore, more effective drugs are needed that spare normal tissue and diminish long-term side effects. Since radiotherapy plays a pivotal role in the treatment of medulloblastoma, we set out to identify novel drugs that could potentiate the effect of ionizing radiation. Thereto, a small molecule library, consisting of 960 chemical compounds, was screened for its ability to sensitize towards irradiation. This small molecule screen identified the flavonoid quercetin as a novel radiosensitizer for the medulloblastoma cell lines DAOY, D283-med, and, to a lesser extent, D458-med at low micromolar concentrations and irradiation doses used in fractionated radiation schemes. Quercetin did not affect the proliferation of neural precursor cells or normal human fibroblasts. Importantly, in vivo experiments confirmed the radiosensitizing properties of quercetin. Administration of this flavonoid at the time of irradiation significantly prolonged survival in orthotopically xenografted mice. Together, these findings indicate that quercetin is a potent radiosensitizer for medulloblastoma cells that may be a promising lead for the treatment of medulloblastoma in patients.

Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves radiation, chemotherapy (temozolomide; TMZ), and maximal surgical resection of the tumor. The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/or acquired resistance to TMZ through MGMT tumor status. Through drug screening, we identified hydroxyurea (HU), an FDA-approved drug, to sensitize GBM cells to TMZ. HU synergized with TMZ in both newly diagnosed GBM with different MGMT status as well as recurrent, TMZ-resistant, tumors in culture as well as in different quantitative intracranial in vivo models. HU acts specifically on the S-phase of the cell cycle by inhibiting the enzyme ribonucleotide reductase M2. Knockdown of this enzyme using RNA interference and other known inhibitors exerted a similar effect to HU in combination with TMZ both in vitro and in vivo. Although HU has been previously evaluated in malignant gliomas in combination with radiation or cytotoxic chemotherapy, and has shown limited efficacy, it was never evaluated in combination with TMZ. In summary, we demonstrate preclinical efficacy of ribonucleotide reductase inhibition using hydroxyurea in combination with TMZ for the treatment of glioblastoma, which warrant further evaluation in a clinical setting.

Sorafenib-a broad kinase inhibitor-is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia-and its consequences on treatment resistance-remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1 alpha) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen-positive (Gr-1(+)) myeloid cell infiltration. The SDF-1 alpha/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1 alpha expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1 alpha increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1(+) myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis-despite persistently elevated hypoxia-in part by reducing Gr-1(+) myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. Conclusion: Blocking SDF-1 alpha/CXCR4 or Gr-1(+) myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment. (Hepatology 2014;59:1435-1447)

Abstract Background: Intrinsic and acquired resistance to temozolomide (TMZ) represents a major obstacle in the treatment of glioblastoma. MicroRNAs (miRNAs) have previously been implicated to play a role in chemoresistance including TMZ resistance. These short non-coding RNA sequences inhibit their mRNA targets, thereby contributing to treatment resistance. In this study, we examined the miRNA/mRNA interaction networks in TMZ resistant glioblastoma cell lines to identify drug targets for the reversal of TMZ resistance. Methods: TMZ resistance was induced in duplicate in the glioblastoma cell lines U87, Hs683, and LNZ308, creating two independent resistant subclones of each wildtype cell line. IC50 values were increased at least two-fold in the TMZ resistant subclones compared to their wildtype. mRNA and miRNA expression profiles of these cell lines were obtained by microarray data analysis. Next, we used these mRNA and miRNA expression profiles of the TMZ resistant cells to generate miRNA/mRNA interaction networks using the integrative network tool mirConnX. This tool combines sequence information with gene expression data analysis to create a condition-specific regulatory network. Results: In the obtained mRNA/miRNA network of our TMZ resistant cell lines, we identified the plant homeodomain (PHD)-like finger 6 (PHF6) mRNA to be targeted by the largest set of miRNAs, i.e. miR-143, miR-93, miR-183, miR-96, and miR-214. Analysis of PHF6 mRNA expression in the TMZ resistant subclones by qRT-PCR showed an increased expression of this mRNA in four of six resistant subclones. Protein expression analysis of PHF6 in these cell lines showed a modest increase in four of the six resistant subclones. Furthermore, analysis of mRNA and protein expression levels of PHF6 in glioblastoma patient datasets showed increased expression of PHF6 in glioblastoma tissue compared to normal brain tissue. In addition, knockdown of PHF6 using siRNA in combination with TMZ enhanced the TMZ response. These results suggest that PHF6 is a candidate drug target for the potentiation of TMZ efficacy. Conclusion: Altogether, these results demonstrate that mirConnX is a useful tool to investigate miRNA/mRNA interactions in TMZ resistant cells and can be used to identify new potential drug targets. Citation Format: Lotte Hiddingh, Rajiv S. Raktoe, Gertjan J.L. Kaspers, W. Peter Vandertop, David P. Noske, Pieter Wesseling, Thomas Wurdinger. Identification of PHF6 as a temozolomide resistance factor in glioblastoma using mirConnX. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2014-3775

Despite the addition of temozolomide (TMZ) to the glioblastoma (GBM) treatment regimen, disease outcome is still poor and additional therapeutic options are necessary. One major obstacle in the GBM treatment is TMZ resistance of the tumor cells resulting in relapse of the disease with no alternative treatments known. In an effort to discover additional treatment modalities we are performing drug screens on GBM cell lines, using wild-type variants and acquired TMZ resistant variants of these cell lines. To be able to screen a range of different cells, we adapted a multiplex bioluminescence reporter assay developed in our lab. This bioluminescent reporter assay is based on the Gaussia luciferase (Gluc) fused to an epitope tag. Different cell lines can be equipped with Gluc fused to different epitope tags (Gluc-tag) so that each cell lines expresses Gluc and a cell line specific epitope tag. Antibody immunoprecipitation allows us to separate the different Gluc-tags and quantify the relative activities of the different cell lines. Activity of each specific Gluc-tag represents the relative number of a specific cell line in the mixed population. Since Gaussia luciferase and the epitope tag is secreted by the cells, we can detect the cell line specific reporters ex vivo in the culture medium or animal blood. Using this novel method in a drug screen we aim to identify additional subtype specific therapy strategies for GBM braintumors. Citation Format: Sjoerd van Rijn, Lotte Hiddingh, Thomas Wurdinger, Ravi Narayan. Drug screen on six co-cultured GBM cell lines using a multiplex luciferase assay. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3727. doi:10.1158/1538-7445.AM2014-3727

Drug resistance is a major issue in the treatment of glioblastoma. Almost all glioblastomas are intrinsically resistant to chemotherapeutic temozolomide (TMZ) or develop resistance during treatment. The interaction networks of microRNAs (miRNAs) and mRNAs likely regulate most biological processes and can be employed to better understand complex processes including drug resistance in cancer. In this study, we examined if integrative miRNA/mRNA network analysis using the web-service tool mirConnX could be used to identify drug resistance factors in glioblastoma. We used TMZ-resistant glioblastoma cells and their integrated miRNA/mRNA networks to identify TMZ-sensitizing factors. TMZ resistance was previously induced in glioblastoma cell lines U87, Hs683, and LNZ308. miRNA/mRNA expression profiling of these cells and integration of the profiles using mirConnX resulted in the identification of plant homeodomain (PHD)-like finger 6 (PHF6) as a potential TMZ-sensitizing factor in resistant glioblastoma cells. Analysis of PHF6 expression showed significant upregulation in glioblastoma as compared to normal tissue. Interference with PHF6 expression in three TMZ-resistant subclones significantly enhanced TMZ-induced cell kill in two of these cell lines. Altogether, these results demonstrate that mirConnX is a feasible and useful tool to investigate miRNA/mRNA interactions in TMZ-resistant cells and has potential to identify drug resistance factors in glioblastoma.

Glioblastoma (GBM) is the most common and most aggressive primary malignant brain tumor. Standard-of-care treatment involves radiation, chemotherapy (temozolomide; TMZ), and maximal surgical resection of the tumor. The 5-year survival rate of patients with GBM is <10%, a colossal failure that has been partially attributed to intrinsic and/ or acquired resistance to TMZ. Here we sought to identify therapeutic agents that could enhance the TMZ effect in GBM cells. Through drug screening, we identified hydroxyurea (HU), an FDA-approved drug, to sensitize GBM cells to TMZ. HU was evaluated as TMZ sensitizer in both newly diagnosed GBM as well as recurrent, TMZ-resistant, tumors. We employed cells obtained from patient tumor tissues with different O6-methylguanine methyl transferase (MGMT) promoter methylation status, which after intracranial injection infiltrate the brain of mice similar to GBM in patients. HU synergized with TMZ in vitro and in vivo, to inhibit tumor growth of both newly diagnosed and recurrent tumors, irrespective of the MGMT promoter methylation status. HU might act specifically on the S-phase of the cell cycle by inhibiting the enzyme ribonucleotide reductase, since knockdown of RRM2 (ribonucleotide reductase M2) exerted a similar effect as HU in combination with TMZ on tumor growth both in vitro and in vivo. Although HU has been previously evaluated in malignant glioma patients in combination with radiation or cytotoxic chemotherapy, and has shown limited efficacy, it was never evaluated in combination with TMZ. Altogether, we demonstrate preclinical efficacy and safety of HU in combination with TMZ for treatment of glioblastoma. These results warrant further evaluation of the combination of HU and TMZ in a clinical setting.

Glioblastoma is the most common malignant primary brain tumor. Temozolomide (TMZ) is the standard chemotherapeutic agent for this disease. However, intrinsic and acquired TMZ-resistance represents a major obstacle for this therapy. In order to identify factors involved in TMZ-resistance, we engineered different TMZ-resistant glioblastoma cell lines. Gene expression analysis demonstrated that EFEMP1, an extracellular matrix protein, is associated with TMZ-resistant phenotype. Silencing of EFEMP1 in glioblastoma cells resulted in decreased cell survival following TMZ treatment, whereas overexpression caused TMZ-resistance. EFEMP1 acts via multiple signaling pathways, including γ-secretase-mediated activation of the Notch pathway. We show that inhibition of γ-secretase by RO4929097 causes at least partial sensitization of glioblastoma cells to temozolomide in vitro and in vivo. In addition, we show that EFEMP1 expression levels correlate with survival in TMZ-treated glioblastoma patients. Altogether our results suggest EFEMP1 as a potential therapeutic target to overcome TMZ-resistance in glioblastoma.

Diffuse intrinsic pontine glioma (DIPG) is a fatal pediatric disease. Thus far, no therapeutic agent has proven beneficial in the treatment of this malignancy. Therefore, conventional DNA-damaging radiotherapy remains the standard treatment, providing transient neurologic improvement without improving the probability of overall survival. During radiotherapy, WEE1 kinase controls the G2 cell-cycle checkpoint, allowing for repair of irradiation (IR)-induced DNA damage. Here, we show that WEE1 kinase is one of the highest overexpressed kinases in primary DIPG tissues compared with matching non-neoplastic brain tissues. Inhibition of WEE1 by MK-1775 treatment of DIPG cells inhibited the IR-induced WEE1-mediated phosphorylation of CDC2, resulting in reduced G2–M arrest and decreased cell viability. Finally, we show that MK-1775 enhances the radiation response of E98-Fluc-mCherry DIPG mouse xenografts. Altogether, these results show that inhibition of WEE1 kinase in conjunction with radiotherapy holds potential as a therapeutic approach for the treatment of DIPG. Mol Cancer Ther; 12(2); 141–50. ©2012 AACR.

Background Glioblastomas exhibit a high level of chemotherapeutic resistance, including to the antimitotic agents vincristine and taxol. During the mitotic agent-induced arrest, glioblastoma cells are able to perform damage-control and self-repair to continue proliferation. Monopolar spindle 1 (MPS1/TTK) is a checkpoint kinase and a gatekeeper of the mitotic arrest.Methods We used glioblastoma cells to determine the expression of MPS1 and to determine the effects of MPS1 inhibition on mitotic errors and cell viability in combination with vincristine and taxol. The effect of MPS1 inhibition was assessed in different orthotopic glioblastoma mouse models (n = 3-7 mice/group). MPS1 expression levels were examined in relation to patient survival.Results Using publicly available gene expression data, we determined that MPS1 overexpression corresponds positively with tumor grade and negatively with patient survival (two-sided t test, P < .001). Patients with high MPS1 expression (n = 203) had a median and mean survival of 487 and 913 days (95% confidence intervals [CI] = 751 to 1075), respectively, and a 2-year survival rate of 35%, whereas patients with intermediate MPS1 expression (n = 140) had a median and mean survival of 858 and 1183 days (95% CI = 1177 to 1189), respectively, and a 2-year survival rate of 56%. We demonstrate that MPS1 inhibition by RNAi results in sensitization to antimitotic agents. We developed a selective small-molecule inhibitor of MPS1, MPS1-IN-3, which caused mitotic aberrancies in glioblastoma cells and, in combination with vincristine, induced mitotic checkpoint override, increased aneuploidy, and augmented cell death. MPS1-IN-3 sensitizes glioblastoma cells to vincristine in orthotopic mouse models (two-sided log-rank test, P < .01), resulting in prolonged survival without toxicity.Conclusions Our results collectively demonstrate that MPS1, a putative therapeutic target in glioblastoma, can be selectively inhibited by MPS1-IN-3 sensitizing glioblastoma cells to antimitotic drugs.

Abstract Brain metastases remain a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)-amplified breast cancer. Unlike HER2-amplified breast tumors growing in extra-cranial locations, brain metastases do not respond well to HER2 inhibitors and are often the reason for treatment failure. One of the major challenges in studying brain metastases is the lack of preclinical models. We developed a HER2-amplified mouse model of brain metastasis using an orthotopic xenograft of BT474 cells in mice. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib failed to contain brain metastatic tumor growth. Based on previous findings from our laboratory suggesting a role of vascular endothelial growth factor (VEGF) in the resistance of HER2-overexpressing breast cancer brain metastases to trastuzumab, we combined HER2 inhibitors with the anti-VEGFR2 antibody DC101. The combination of either trastuzumab and DC101 or lapatinib and DC101 significantly slowed metastatic tumor growth in the brain, and resulted in a striking improvement in overall survival. The benefit is due largely to an anti-angiogenic effect. The combination of anti-HER2 and anti-VEGFR2 therapy reduced both the total and functional microvascular density in the brain metastatic tumors. Moreover, tumor tissues under combination therapy showed a marked increase in necrosis. Preclinical and clinical evidence suggest that the combination of trastuzumab and lapatinib is superior to either agent alone – though this has never been tested in the brain metastatic setting. We consistently observed increased phosphorylation of HER2 in breast tumor cells growing in the brain compared with the mammary fat pad. In addition, while short-term lapatinib treatment significantly reduced HER2 activation in the brain, it could do so only to the level of that observed in the untreated mammary fat pad - and this effect disappeared over time. We hypothesized that more pronounced HER2 inhibition would be beneficial to these brain metastases with increased HER2 activation. We show here a significant growth delay with the combination of the two HER2 inhibitors compared with monotherapy. Moreover, we found a dramatic brain metastatic tumor growth delay in mice treated with both HER2 inhibitors, trastuzumab and lapatinib, and DC101. The triple combination prolonged overall survival 5 times longer than control-treated mice. Brain metastasis from breast cancer is considered the “final frontier” of breast cancer research and treatment. Our findings support the clinical development of a three-drug regimen of trastuzumab, lapatinib and a VEGF pathway inhibitor for the treatment of HER2-amplified breast cancer brain metastases. While the anti-VEGF antibody bevacizumab in combination with trastuzumab and chemotherapy has shown some promise in HER2-positive metastatic breast cancer patient, there are no data on its efficacy in the context of brain metastases. A clinical trial is now recruiting patients to evaluate the efficacy of bevacizumab in breast cancer patients with active brain metastases, including its combination with trastuzumab in patients with HER2-positive disease. This trial may provide clinical evidence for the approach presented here. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-12-03.

Targeting dendritic cells (DC) through the release of suppressive factors is an effective means for tumors to escape immune control. We assessed the involvement of downstream signaling through the JAK2/STAT3 and p38 MAPK pathways in tumor-induced suppression of human DC development. Whereas the JAK2/STAT3 pathway has been pinpointed in mouse studies as a key regulator of myeloid suppression, in human DC this is less well established. We studied the effects of STAT3 inhibition on the suppression of monocyte-derived DC differentiation mediated by a short-list of four predominant suppressive factors and found that pharmacological STAT3 inhibition could only counteract the effects of IL-6. Accordingly, in testing a panel of supernatants derived from 11 cell lines representing various types of solid tumors, STAT3 inhibition only modestly affected the suppressive effects of a minority of supernatants. Importantly, combined interference in the STAT3 and p38 pathways completely prevented inhibition of DC differentiation by all tested supernatants and effected superior DC function, evidenced by increased allogeneic T cell reactivity with elevated IL-12p70/IL-10 ratios and Th1 skewing. Combined STAT3 and p38 inhibition also afforded superior protection against the suppressive effects of primary glioma and melanoma supernatants and induced a shift from CD14+ cells to CD1a+ cells in metastatic melanoma single-cell suspensions, indicating a potential for improved DC differentiation in the tumor microenvironment. We conclude that combined interference in the STAT3 and p38 MAPK signaling pathways is a promising approach to overcome tumor-induced inhibitory signaling in DC precursors and will likely support clinical immunotherapeutic strategies.

Brain metastases are a serious obstacle in the treatment of patients withhuman epidermal growth factor receptor-2(HER2)–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model ofHER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment ofHER2-amplified breast cancer brain metastases.