Gliomas represent approximately 30% of all central nervous system tumors and 80% of malignant brain tumors1. To understand the molecular mechanisms underlying the malignant progression of low-grade gliomas with mutations in IDH1 (encoding isocitrate dehydrogenase 1), we studied paired tumor samples from 41 patients, comparing higher-grade, progressed samples to their lower-grade counterparts. Integrated genomic analyses, including whole-exome sequencing and copy number, gene expression and DNA methylation profiling, demonstrated nonlinear clonal expansion of the original tumors and identified oncogenic pathways driving progression. These include activation of the MYC and RTK-RAS-PI3K pathways and upregulation of the FOXM1- and E2F2-mediated cell cycle transitions, as well as epigenetic silencing of developmental transcription factor genes bound by Polycomb repressive complex 2 in human embryonic stem cells. Our results not only provide mechanistic insight into the genetic and epigenetic mechanisms driving glioma progression but also identify inhibition of the bromodomain and extraterminal (BET) family as a potential therapeutic approach.

Acute lymphoblastic leukaemia (ALL) has a marked propensity to metastasize to the central nervous system (CNS). In contrast to brain metastases from solid tumours, metastases of ALL seldom involve the parenchyma but are isolated to the leptomeninges, which is an infrequent site for carcinomatous invasion. Although metastasis to the CNS occurs across all subtypes of ALL, a unifying mechanism for invasion has not yet been determined. Here we show that ALL cells in the circulation are unable to breach the blood-brain barrier in mice; instead, they migrate into the CNS along vessels that pass directly between vertebral or calvarial bone marrow and the subarachnoid space. The basement membrane of these bridging vessels is enriched in laminin, which is known to coordinate pathfinding of neuronal progenitor cells in the CNS. The laminin receptor α6 integrin is expressed in most cases of ALL. We found that α6 integrin-laminin interactions mediated the migration of ALL cells towards the cerebrospinal fluid in vitro. Mice with ALL xenografts were treated with either a PI3Kδ inhibitor, which decreased α6 integrin expression on ALL cells, or specific α6 integrin-neutralizing antibodies and showed significant reductions in ALL transit along bridging vessels, blast counts in the cerebrospinal fluid and CNS disease symptoms despite minimally decreased bone marrow disease burden. Our data suggest that α6 integrin expression, which is common in ALL, allows cells to use neural migratory pathways to invade the CNS.

Previous studies have shown that polycyclic aromatic hydrocarbons (PAHs) mobilize intracellular Ca2+ in human T cells by inositol trisphosphate-dependent mechanisms resulting from activation of phospholipase C-gamma by SRC-related protein tyrosine kinases, thereby mimicking antigen-receptor activation. Ca2+ appears to play an important second messenger role in growth factor control of cell proliferation in human mammary epithelial cells (HMEC), such as the epidermal growth factor receptor pathway. The purpose of the present studies was to determine if PAHs are able to increase intracellular Ca2+ in primary cultures of HMEC and increase cell proliferation. Two carcinogenic and two non-carcinogenic PAHs were tested for their ability to increase intracellular Ca2+ in HMEC. The carcinogenic PAHs dimethylbenz[a]anthracene (DMBA) and benzo[a] pyrene (BaP) were able to cause Ca2+ elevation in HMEC at early time points (2 h) and caused sustained alterations in Ca2+ homeostasis (18 h). DMBA showed maximal effects at early time points (2 h), while BaP showed maximal effects on sustained Ca2+ (18 h). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a potent dioxin and tumor promoter, produced maximal Ca2+ elevation at 2 h, with a return to near baseline levels by 6 h. The non-carcinogenic PAHs benzo[e]pyrene and anthracene did not significantly alter intracellular Ca2+ at any time point. alpha-Naphthoflavone significantly reduced the Ca2+ response induced by BaP treatment, but not by DMBA or TCDD, suggesting that P450 1A or 1B metabolism of BaP may be important in the sustained Ca2+ elevating response. In evaluating the effects of BaP on HMEC proliferation, BaP was found to increase the number of cells recovered after 4 days in culture in the absence or presence of various concentrations of epidermal growth factor. These studies provide initial evidence that Ca2+ signaling may be associated with mitogenesis in HMEC, which may play a role in tumor promotion and progression produced by PAHs.

The fibroblast growth factor receptor (FGFR)2 gene has been shown to be amplified in 5-10% of breast cancer patients. A breast cancer cell line developed in our laboratory, SUM-52PE, was shown to have a 12-fold amplification of the FGFR2 gene, and FGFR2 message was found to be overexpressed 40-fold in SUM-52PE cells as compared with normal human mammary epithelial (HME) cells. Both human breast cancer (HBC) cell lines and HME cells expressed two FGFR2 isoforms, whereas SUM-52PE cells overexpressed those two isoforms, as well as several unique FGFR2 polypeptides. SUM-52PE cells expressed exclusively FGFR2-IIIb isoforms, which are high-affinity receptors for fibroblast growth factor (FGF)-1 and FGF-7. Differences were identified in the expression of the extracellular Ig-like domains, acid box and carboxyl termini, and several variants not previously reported were isolated from these cells. The electronic version of this article can be found online at http://breast-cancer-research.com/content/2/4/311 © Current Science Ltd FGF = fibroblast growth factor; FGFR = fibroblast growth factor receptor; KGF = keratinocyte growth factor; HME = human mammary epithelial (cell); HBC = human breast cancer (cell); PCR = polymerase chain reaction; RT = reverse transcription. Introduction:The FGFR family of receptor tyrosine kinases includes four members, all of which are highly alternatively spliced and glycosylated. For FGFR2, alternative splicing of the second half of the third Ig-like domain, involving exons IIIb and IIIc, is a mutually exclusive choice that affects ligand binding specificity and affinity [1][2][3]. It appears that the second half of the third Ig-like domain can dictate high affinity for FGF-2 or keratinocyte growth factor (KGF), whereas affinity for FGF-1 appears to remain the same [3]. Alternative splicing of the carboxyl terminus has been shown to involve at least two different exons that can produce at least three different variants. The C1-type and C2-type carboxyl termini are encoded by the same exon, and have two different splice acceptor sites, whereas the C3-type carboxyl terminus is encoded by a separate exon [4]. The biologic significance of the C1 carboxyl terminus, as compared with the shorter C3 variant found primarily in tumorigenic samples, has been studied in NIH3T3 transfection assays, in which C3 variants were able to produce three times more transformed foci in soft agar than C1 variants (both IIIb), whereas full length FGFR2 and FGFR1 (both IIIc variants) showed no transforming activity [4]. Previous studies [5,6] have found amplification and overexpression of FGFR2 in 5-10% of primary breast cancer specimens. A recent study [7] done using a tissue array consisting of 372 primary breast cancer specimens found a 5% incidence of FGFR2 amplification. To our knowledge, none of the HBC cell lines studied thus far have an FGFR2 gene amplification, although overexpression of FGFR2

Previous studies in this laboratory have shown that polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP), and certain halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), modulate receptor signaling pathways in human lymphoid and non-lymphoid cells. We have recently demonstrated that BaP produces a weak mitogenic signal in human mammary epithelial cells, perhaps by mimicking growth factor signaling pathways. In the present studies we found that BaP and TCDD activated insulin-like growth factor (IGF-I) signaling pathways under insulin-deficient conditions. The effects of BaP and TCDD were evaluated in the human MCF-10A mammary epithelial cell line grown under epidermal growth factor- and insulin-dependent conditions. BaP (0.3 microM) and TCDD (30 nM) were found to restore a moderate insulin-like signal in MCF-10A cells grown in the absence of added insulin. TCDD was more potent and produced better activation of cell growth than did BaP. Both TCDD and BaP appeared to mimic signaling through the IGF-I receptor (IGF-IR), as evidenced by increased tyrosine phosphophorylation of IGF-IRbeta, IRS-1 and Shc. In addition, both BaP and TCDD significantly increased the activity of phosphatidylinositol 3-kinase (PI3K). The PI3K inhibitor LY294002 was found to inhibit the growth-promoting effects of TCDD seen under insulin-deficient conditions. The results of these studies show that under certain conditions BaP and TCDD can mimic growth factor signaling pathways in human mammary epithelial cells, demonstrating that environmentally prevalent carcinogenic compounds may alter cell growth in human mammary epithelial cells via mimicry of growth factor receptor signaling pathways.

Targeted therapy is revolutionizing the treatment of cancers, but resistance evolves against these therapies and derogates their success. The phosphatidylinositol 3-kinase delta (PI3K-δ) inhibitor idelalisib has been approved for treatment of chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma, but the mechanisms conferring resistance in a subset of patients are unknown. Here, we modeled resistance to PI3K-δ inhibitor in vivo using a serial tumor transfer and treatment scheme in mice. Whole-exome sequencing did not identify any recurrent mutation explaining resistance to PI3K-δ inhibitor. In the murine model, resistance to PI3K-δ inhibitor occurred as a result of a signaling switch mediated by consistent and functionally relevant activation of insulin-like growth factor 1 receptor (IGF1R), resulting in enhanced MAPK signaling in the resistant tumors. Overexpression of IGF1R in vitro demonstrated its prominent role in PI3K-δ inhibitor resistance. IGF1R upregulation in PI3K-δ inhibitor–resistant tumors was mediated by functional activation and enhanced nuclear localization of forkhead box protein O1 transcription factors and glycogen synthase kinase 3β. In human CLL, high IGF1R expression was associated with trisomy 12. CLL cells from an idelalisib-treated patient showed decreased sensitivity to idelalisib in vitro concomitant with enhanced MAPK signaling and strong upregulation of IGF1R upon idelalisib exposure. Thus, our results highlight that alternative signaling cascades play a predominant role in the resistance and survival of cancer cells under PI3K-δ inhibition. We also demonstrate that these pathway alterations can serve as therapeutic targets, because inhibition of IGF1R offered efficacious salvage treatment of PI3K-δ inhibitor–resistant tumors in vitro and in vivo.

Activated B-cell-like diffuse large B-cell lymphoma relies on B-cell receptor signaling to drive proliferation and survival. Downstream of the B-cell receptor, the key signaling kinases Bruton’s tyrosine kinase and phosphoinositide 3-kinase δ offer opportunities for therapeutic intervention by agents such as ibrutinib, ONO/GS-4059, and idelalisib. Combination therapy with such targeted agents could provide enhanced efficacy due to complimentary mechanisms of action. In this study, we describe both the additive interaction of and resistance mechanisms to idelalisib and ONO/GS-4059 in a model of activated B-cell-like diffuse large B-cell lymphoma. Significant tumor regression was observed with a combination of PI3Kδ and Bruton’s tyrosine kinase inhibitors in the mouse TMD8 xenograft. Acquired resistance to idelalisib in the TMD8 cell line occurred by loss of phosphatase and tensin homolog and phosphoinositide 3-kinase pathway upregulation, but not by mutation of PIK3CD. Sensitivity to idelalisib could be restored by combining idelalisib and ONO/GS-4059. Further evaluation of targeted inhibitors revealed that the combination of idelalisib and the phosphoinositide-dependent kinase-1 inhibitor GSK2334470 or the AKT inhibitor MK-2206 could partially overcome resistance. Characterization of acquired Bruton’s tyrosine kinase inhibitor resistance revealed a novel tumor necrosis factor alpha induced protein 3 mutation (TNFAIP3 Q143*), which led to a loss of A20 protein, and increased p-IκBα. The combination of idelalisib and ONO/GS-4059 partially restored sensitivity in this resistant line. Additionally, a mutation in Bruton’s tyrosine kinase at C481F was identified as a mechanism of resistance. The combination activity observed with idelalisib and ONO/GS-4059, taken together with the ability to overcome resistance, could lead to a new therapeutic option in activated B-cell-like diffuse large B-cell lymphoma. A clinical trial is currently underway to evaluate the combination of idelalisib and ONO/GS-4059 (NCT02457598).

Despite idelalisib approval in relapsed follicular lymphoma (FL), a complete characterization of the immunomodulatory consequences of phosphatidylinositol 3-kinase δ (PI3Kδ) inhibition, biomarkers of response, and potential combinatorial therapies in FL remain to be established. Using ex vivo cocultures of FL patient biopsies and follicular dendritic cells (FDCs) to mimic the germinal center (n = 42), we uncovered that PI3Kδ inhibition interferes with FDC-induced genes related to angiogenesis, extracellular matrix formation, and transendothelial migration in a subset of FL samples, defining an 18-gene signature fingerprint of idelalisib sensitivity. A common hallmark of idelalisib found in all FL cases was its interference with the CD40/CD40L pathway and induced proliferation, together with the downregulation of proteins crucial for B–T-cell synapses, leading to an inefficient cross talk between FL cells and the supportive T-follicular helper cells (TFH). Moreover, idelalisib downmodulates the chemokine CCL22, hampering the recruitment of TFH and immunosupressive T-regulatory cells to the FL niche, leading to a less supportive and tolerogenic immune microenvironment. Finally, using BH3 profiling, we uncovered that FL–FDC and FL–macrophage cocultures augment tumor addiction to BCL-XL and MCL-1 or BFL-1, respectively, limiting the cytotoxic activity of the BCL-2 inhibitor venetoclax. Idelalisib restored FL dependence on BCL-2 and venetoclax activity. In summary, idelalisib exhibits a patient-dependent activity toward angiogenesis and lymphoma dissemination. In all FL cases, idelalisib exerts a general reshaping of the FL immune microenvironment and restores dependence on BCL-2, predisposing FL to cell death, providing a mechanistic rationale for investigating the combination of PI3Kδ inhibitors and venetoclax in clinical trials.