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Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease. Significance: A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunologic changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+, and CD8+ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM-bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma. Significance: Microparticle-mediated local inhibition of NAMPT modulates the tumor immune microenvironment and acts cooperatively with anti-PD-1 checkpoint blockade, offering a combination immunotherapy strategy for the treatment of GBM.

The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunological changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+, and CD8+ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma.

The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD

IDH1-mutant gliomas are dependent upon the canonical coenzyme NAD+ for survival. It is known that PARP activation consumes NAD+ during base excision repair (BER) of chemotherapy-induced DNA damage. We therefore hypothesized that a strategy combining NAD+ biosynthesis inhibitors with the alkylating chemotherapeutic agent temozolomide could potentiate NAD+ depletion–mediated cytotoxicity in mutant IDH1 cancer cells. To investigate the impact of temozolomide on NAD+ metabolism, patient-derived xenografts and engineered mutant IDH1-expressing cell lines were exposed to temozolomide, in vitro and in vivo, both alone and in combination with nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which block NAD+ biosynthesis. The acute time period (

The aggressive primary brain tumor glioblastoma (GBM) is characterized by aberrant metabolism that fuels its malignant phenotype. Diverse genetic subtypes of malignant glioma are sensitive to selective inhibition of the NAD+ salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT). However, the potential impact of NAD+ depletion on the brain tumor microenvironment has not been elaborated. In addition, systemic toxicity of NAMPT inhibition remains a significant concern. Here we show that microparticle-mediated intratumoral delivery of NAMPT inhibitor GMX1778 induces specific immunologic changes in the tumor microenvironment of murine GBM, characterized by upregulation of immune checkpoint PD-L1, recruitment of CD3+, CD4+, and CD8+ T cells, and reduction of M2-polarized immunosuppressive macrophages. NAD+ depletion and autophagy induced by NAMPT inhibitors mediated the upregulation of PD-L1 transcripts and cell surface protein levels in GBM cells. NAMPT inhibitor modulation of the tumor immune microenvironment was therefore combined with PD-1 checkpoint blockade in vivo, significantly increasing the survival of GBM-bearing animals. Thus, the therapeutic impacts of NAMPT inhibition extended beyond neoplastic cells, shaping surrounding immune effectors. Microparticle delivery and release of NAMPT inhibitor at the tumor site offers a safe and robust means to alter an immune tumor microenvironment that could potentiate checkpoint immunotherapy for glioblastoma. SIGNIFICANCE: Microparticle-mediated local inhibition of NAMPT modulates the tumor immune microenvironment and acts cooperatively with anti-PD-1 checkpoint blockade, offering a combination immunotherapy strategy for the treatment of GBM.

Using a three-dimensional coculture model, we identified significant subtype-specific changes in gene expression, metabolic, and therapeutic sensitivity profiles of breast cancer cells in contact with cancer-associated fibroblasts (CAF). CAF-induced gene expression signatures predicted clinical outcome and immune-related differences in the microenvironment. We found that fibroblasts strongly protect carcinoma cells from lapatinib, attributable to its reduced accumulation in carcinoma cells and an elevated apoptotic threshold. Fibroblasts from normal breast tissues and stromal cultures of brain metastases of breast cancer had similar effects as CAFs. Using synthetic lethality approaches, we identified molecular pathways whose inhibition sensitizes HER2+ breast cancer cells to lapatinib both in vitro and in vivo, including JAK2/STAT3 and hyaluronic acid. Neoadjuvant lapatinib therapy in HER2+ breast tumors lead to a significant increase of phospho-STAT3+ cancer cells and a decrease in the spatial proximity of proliferating (Ki67+) cells to CAFs impacting therapeutic responses. Our studies identify CAF-induced physiologically and clinically relevant changes in cancer cells and offer novel approaches for overcoming microenvironment-mediated therapeutic resistance. Cancer Res; 76(22); 6495–506. ©2016 AACR.

Although lung adenocarcinomas frequently metastasize to the brain, treatment options for lung adenocarcinoma brain metastases (BM-LUAD) are limited. We discovered novel candidate drivers of progression by using case-control analyses to compare whole-exome sequencing data from a cohort of 73 BM-LUAD to a control cohort of 503 primary lung adenocarcinomas. We identified MYC, YAP1 and MMP13 as genomic regions with significantly more frequent amplifications in BM-LUAD compared to control cohort. We validated that MYC, YAP1 and MMP13 can drive brain metastases in a patient-derived xenograft mouse model, where incidence of brain metastases was higher in mice injected with tumor cells expressing the candidate drivers compared to tumor cells expressing LacZ. These results indicate that somatic alterations can drive lung adenocarcinomas to metastasize to the brain. These candidate drivers may serve as therapeutic targets in patients with brain metastatic lung adenocarcinomas. Citation Format: Naema Nayyar, David J. Shih, Ivanna Bihun, Ibiayi Dagogo-Jack, Corey M. Gill, Elisa Aquilanti, Mia Bertalan, Alexander Kaplan, Megan R. D'Andrea, Ugonma Chukwueke, Christopher Alvarez-Breckenridge, Matthew Lastrapes, Ben Kuter, Matthew R. Strickland, Juan Carlos Martinez-Gutierrez, Deepika Nagabhushan, Magali De Sauvage, Michael D. White, Brandyn A. Castro, Kaitlin Hoang, Sun Ha Paek, Sun Hye Park, Maria Martinez-Lage, Anna S. Berghoff, Parker Merrill, Elizabeth R. Gerstner, Tracy T. Batchelor, Matthew P. Frosch, Ryan P. Frazier, Darrell R. Borger, A John Iafrate, Sandro Santagata, Matthias Preusser, Daniel P. Cahill, Scott L. Carter, Priscilla K. Brastianos. Identifying genomic drivers of lung adenocarcinoma brain metastases [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4729.

IDH1 -mutant gliomas are dependent upon the canonical coenzyme nicotinamide adenine dinucleotide (NAD+) for survival. It is known that Poly(ADP-ribose) polymerase (PARP) activation consumes NAD+ during base excision repair (BER) of chemotherapy-induced DNA damage. We therefore hypothesized that a strategy combining NAD+ biosynthesis inhibitors with the alkylating chemotherapeutic agent temozolomide (TMZ) could potentiate NAD+ depletion-mediated cytotoxicity in mutant IDH1 cancer cells. To investigate the impact of TMZ on NAD+ metabolism, patient-derived xenografts and engineered mutant IDH1-expressing cell lines were exposed to TMZ, in vitro and in vivo, both alone and in combination with nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which block NAD+ biosynthesis. The acute time-period (

Background: Mismatch repair (MMR) deficiency through MSH6 alteration has been identified in approximately 30% of recurrent high grade gliomas, and represents a key molecular mechanism for the development of acquired resistance to the alkylating agent temozolomide (TMZ). Additionally, glioma progression post-TMZ treatment is frequently accompanied by distinct genomic alterations such as amplification of the MYC locus and resulting signaling activation. There is an urgent need to establish new treatment strategies that do not rely upon MMR to target MMR deficient, TMZ resistant glioma. Methods: We first screened 14 compounds that modulate DNA damage response for their ability to suppress viability of an MSH6 knockdown, TMZ resistant glioma cell line. Efficacy of PLK1 inhibitors and combination with TMZ were assessed in glioblastoma cells lines and patient-derived glioblastoma neurosphere lines engineered with MSH6 silencing, MYC overexpression and controls. Impacts of PLK1 blockade on cell cycle distribution, apoptosis, and DNA damage effects were examined. Results: The compound screening indentified PLK1 selective inhibitor, volasertib, as the most potent inhibitor of proliferation of both MMR intact and deficient glioblastoma cells. PLK1 inhibition induced G2/M cell cycle arrest, DNA damage and caspase-mediated apoptosis in glioblastoma cells, but no detectable toxicity in normal human astrocytes. Importantly, beneficial therapeutic effects of PLK1 inhibitors were not influenced by MMR deficiency due to MSH6 knockdown, whereas PLK1 inhibition had no additive or synergic effect when combined with TMZ. Furthermore, we found that MYC overexpression sensitized glioblastoma cells to PLK1 inhibitors. Conclusions: Selective PLK1 inhibitors are potently cytotoxic to glioblastoma and their action is independent of MMR status of the cells. Cells with deregulated MYC are vulnerable to PLK1 inhibition suggesting genomic MYC alteration as a biomarker for PLK inhibitor sensitivity. Thus PLK inhibitor represents a novel therapeutic option for MMR deficient TMZ resistant recurrent gliomas, particularly those driven by MYC. Citation Format: Fumi Higuchi, Daniel P. Cahill, Hiroaki Wakimoto. PLK1 inhibitor targets mismatch repair-deficient temozolomide-resistant tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-301. doi:10.1158/1538-7445.AM2017-LB-301

Background: Brain metastases represent an unmet need in current oncologic care. Approximately 8-10% of cancer patients will develop brain metastases, and more than half of these patients will pass away within a few months of their diagnosis. We have a limited understanding of how brain metastases genetically evolve from their primary tumors. Our objectives were to (1) elucidate the genomic evolutionary patterns leading to brain metastases (2) identify whether brain metastases harbor clinically significant genetic differences compared to their primary tumors and other extracranial metastatic sites, and (3) examine the extent of genetic heterogeneity across regionally separated and anatomically distinct sites of brain metastasis. Methods: We subjected 104 matched primary tumor biopsies, brain metastases, and normal tissue to whole exome sequencing, including 20 cases with regionally and anatomically separated brain metastasis sites, regional lymph nodes, and distal extracranial metastases. We performed an integrative analysis of somatic single nucleotide variants and copy-number alterations to reconstruct phylogenetic trees relating the subclones from each patient. We analyzed evolutionary relationships between related cancer samples and annotated phylogenetic trees with clinically significant genetic alterations. Results: Every brain metastasis displayed branched evolution: the brain metastasis and primary tumor shared a common ancestor yet both the primary tumor and brain metastasis continued to evolve independently. We found novel clinically actionable genetic alterations that were exclusive to brain metastases in 56% of cases. The brain metastases were also enriched for several pathways, some pathways specific to a particular histology. Distal extracranial metastases and regional lymph nodes were highly divergent from brain metastases, and in no cases, did we observe an extracranial site that closely resembled the brain metastasis. In contrast, regionally and anatomically separated brain metastasis sites were genetically homogenous and shared nearly all genetic alterations detected. Conclusions: Brain metastases are genetically divergent from primary tumors. Clinically, these observations demonstrate that biopsies of primary tumors fail to capture the heterogeneity with patients with brain metastases, potentially missing clinically actionable mutations in these life-threatening metastases. Notably, regional lymph nodes and distal extracranial metastases were not reliable genetic surrogates for brain metastases. When clinically feasible, characterization of even a single brain metastasis lesion is superior to that of a primary or lymph node biopsy for selection of a targeted therapeutic agent. Citation Format: Priscilla K. Brastianos, Scott L. Carter, Sandro Santagata, Amaro Taylor-Weiner, Robert T. Jones, Eli Van Allen, Keith L. Ligon, Josep Tabernero, Joan Seoane, Elena Martinez-Saez, Daniel Cahill, William T. Curry, Ian F. Dunn, Sun Ha Paek, Paul Van Hummelen, Aaron R. Thorner, Bruce E. Johnson, Nancy U. Lin, Toni K. Choueiri, Michael S. Rabin, Rameen Beroukhim, Anat Stemmer-Rachamimov, Matthew Meyerson, Levi Garraway, Tracy Batchelor, Jose Baselga, David N. Louis, William C. Hahn, Gad Getz. Genomic characterization of brain metastases reveals divergent evolution and metastasis specific mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4727. doi:10.1158/1538-7445.AM2015-4727

Diffuse gliomas are up till now graded based upon morphology. Recent findings indicate that isocitrate dehydrogenase (IDH) mutation status defines biologically distinct groups of tumors. The role of tumor grade and mitotic index in patient outcome has not been evaluated following stratification by IDH mutation status. To address this, we interrogated 558 WHO grade II-III diffuse gliomas for IDH1/2 mutations and investigated the prognostic impact of WHO grade within IDH-mutant and wild-type tumor subsets independently. The prognostic impact of grade was modest in IDH-mutant [hazard ratio (HR) = 1.21, 95% confidence interval (CI) = 0.91-1.61] compared to IDH-wild type tumors (HR = 1.74, 95% CI = 0.95-3.16). Using a dichotomized mitotic index cut-off of 4/1000 tumor cells, we found that while mitotic index was significantly associated with outcome in IDH-wild type tumors (log-rank p Citation Format: Adriana Olar, Khalida Wani, Kristin Diefes, Lindsey Heathcock, Hinke van Thuijl, Mark Gilbert, Terri Armstrong, Erik Sulman, Daniel Cahill, Jaap Reijneveld, Bauke Ylstra, Pieter Wesseling, Kenneth Aldape. IDH mutation status and role of WHO grade and mitotic index in overall survival in grade II-III diffuse gliomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5270. doi:10.1158/1538-7445.AM2015-5270

Although most colorectal cancers are chromosomally unstable, the basis for this instability has not been defined. To determine whether genes shown to cause chromosomal instability in model systems were mutated in colorectal cancers, we identified their human homologues and determined their sequence in a panel of colorectal cancers. We found 19 somatic mutations in five genes representing three distinct instability pathways. Seven mutations were found in MRE11, whose product is involved in double-strand break repair. Four mutations were found among hZw10, hZwilch/FLJ10036, and hRod/KNTC, whose products bind to one another in a complex that localizes to kinetochores and controls chromosome segregation. Eight mutations were found in Ding, a previously uncharacterized gene with sequence similarity to the Saccharomyces cerevisiae Pds1, whose product is essential for proper chromosome disjunction. This analysis buttresses the evidence that chromosomal instability has a genetic basis and provides clues to the mechanistic basis of instability in cancers.

Background: Brain metastases are the most frequently occurring intracranial tumors in adults. Median survival after the diagnosis of a brain metastasis is in the order of a few months. Despite its large burden of disease and devastating clinical sequelae, we continue to have a limited understanding of how brain metastases evolve from their primary tumor. Our objectives were to (1) elucidate the evolutionary patterns leading the brain metastases and (2) identify whether brain metastases are genetically distinct from their matched primary tumors. Materials and Methods: We subjected 101 trios consisting of primary tumor, brain metastasis, and matched normal tissue to whole exome sequencing (WES). To analyze the data, we developed novel computational tools to perform an integrative analysis of somatic single nucleotide variants (SSNVs) and somatic copy-number alterations (SCNAs). This analysis allowed us to estimate the clonal architecture of the primary and metastatic samples from each patient, and to reconstruct a phylogenetic tree relating all of the subclones.Results: Every metastasis developed from a single clone, consistent with a single cell of origin. We did not detect evidence of self-seeding or a multiclonal origin of metastasis. In all cases, we observed a sibling or a branched evolutionary relationship; the brain metastasis and primary tumor share a common ancestor, but there was continued evolution in the primary tumor reflected by fully clonal mutations in the primary biopsy that were not present in the metastasis. When we average over all the phylogenetic trees, 61% of mutations are present in the common ancestor, 24% are unique to the metastasis and 15% are unique to the primary tumor. Subclonal mutations in the metastasis by definition occurred within the brain; these mutations displayed different mutational signatures than those acquired in the primary tumor. These contrasts were most pronounced in cases of lung cancer or melanoma, with tobacco and UV signatures prominent in these primaries and nearly absent from the mutations acquired after metastasis. In order to understand the molecular drivers of clonal evolution and metastasis in our data, we annotated each subclone with driver mutations identified using large numbers of cancer samples analyzed by the cancer genome atlas (TCGA) consortium. This produced a detailed portrait of each patient's cancer, with nearly node in each phylogenetic tree associated with at least one known driver. We found novel drivers, many of which are known actionable targets, in the clonal and subclonal populations within the brain metastases that were not present in the primary tumor. This suggests ongoing evolution within the brain. Similarly, novel subclonal and clonal drivers were detected in the biopsy of the primary tumor that were not present in the metastasis. The brain metastases were enriched for several pathways when compared to their matched primary tumors, some pathways specific to a particular histologic subtype.Conclusions: In this study, we report the latest results of the largest massively parallel sequencing study to date of matched brain metastases and primary tumors. We used intratumoral heterogeneity estimates to elucidate the evolutionary patterns observed in the process of metastasis. This study shifts our understanding of the metastasis paradigm and sheds light on the evolutionary and molecular mechanisms that are critical for brain metastasis. Our data suggests that single biopsies do not capture the heterogeneity within patients. Assessment of the subclonal phylogenetic architecture of primaries and their metastases should be considered when selecting targeted agents for patients with brain metastases. Citation Format: Priscilla K. Brastianos, Scott L. Carter, Sandro Santagata, Amaro Taylor-Weiner, Robert T. Jones, Peleg M. Horowitz, Keith L. Ligon, Joan Seaone, Elena Martinez-Saez, Josep Tabernero, Daniel P. Cahill, Sun-Ha Paek, Ian F. Dunn, Bruce E. Johnson, Toni K. Choueiri, Michael S. Rabin, Eric P. Winer, Nancy U. Lin, Paul Van Hummelen, Anat Stemmer-Rachamimov, Rameen Beroukhim, David L. Louis, Tracy T. Batchelor, Jose Baselga, Gad Getz, William C. Hahn. Genomic characterization of 101 brain metastases and paired primary tumors reveals patterns of clonal evolution and selection of driver mutations. [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 NG03. doi:10.1158/1538-7445.AM2014-NG03