Flor Mendez

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

The loss of ATRX impairs DNA repair, promoting glioma growth but enhancing sensitivity to DNA-damaging therapies. Aggressive gliomas’ Achilles’ heel ATRX is a protein that is often mutated in glioma, a lethal and relatively common brain tumor. Koschmann et al. developed a mouse model of ATRX-deficient glioma and discovered that these tumors grow more aggressively than their counterparts with wild-type ATRX. The reason this happens is that the loss of ATRX impairs DNA repair, resulting in genetically unstable tumors that can accumulate oncogenic mutations more quickly. However, because of their DNA repair defect, these tumors also proved to be more sensitive to treatments that damage the DNA, such as radiation and some types of chemotherapy. Consistent with these findings, the presence of ATRX mutation correlated with better outcomes in patients, because these tumors were more susceptible to treatment. Recent work in human glioblastoma (GBM) has documented recurrent mutations in the histone chaperone protein ATRX. We developed an animal model of ATRX-deficient GBM and showed that loss of ATRX reduces median survival and increases genetic instability. Further, analysis of genome-wide data for human gliomas showed that ATRX mutation is associated with increased mutation rate at the single-nucleotide variant (SNV) level. In mouse tumors, ATRX deficiency impairs nonhomologous end joining and increases sensitivity to DNA-damaging agents that induce double-stranded DNA breaks. We propose that ATRX loss results in a genetically unstable tumor, which is more aggressive when left untreated but is more responsive to double-stranded DNA-damaging agents, resulting in improved overall survival.

Myeloid cells are required for PD-1/PD-L1 checkpoint activation and the establishment of an immunosuppressive environment in pancreatic cancer

Background Pancreatic cancer is characterised by the accumulation of a fibro-inflammatory stroma. Within this stromal reaction, myeloid cells are a predominant population. Distinct myeloid subsets have been correlated with tumour promotion and unmasking of anti-tumour immunity. Objective The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. Methods Primary mouse pancreatic cancer cells were transplanted into CD11b-diphtheria toxin receptor (DTR) mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells (mostly myeloid cell population) were depleted by diphtheria toxin treatment during tumour initiation or in established tumours. Results Depletion of myeloid cells prevented KrasG12D-driven pancreatic cancer initiation. In pre-established tumours, myeloid cell depletion arrested tumour growth and in some cases, induced tumour regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T-cell anti-tumour activity by inducing the expression of programmed cell death-ligand 1 (PD-L1) in tumour cells in an epidermal growth factor receptor (EGFR)/mitogen-activated protein kinases (MAPK)-dependent manner. Conclusion Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK-dependent regulation of PD-L1 expression on tumour cells. Derailing this crosstalk between myeloid cells and tumour cells is sufficient to restore anti-tumour immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer.

Mutated Chromatin Regulatory Factors as Tumor Drivers in Cancer

Genes encoding proteins that regulate chromatin structure and DNA modifications [i.e., chromatin regulatory factors (CRF)] and genes encoding histone proteins harbor recurrent mutations in most human cancers. These mutations lead to modifications in tumor chromatin and DNA structure and an altered epigenetic state that contribute to tumorigenesis. Mutated CRFs have now been identified in most types of cancer and are increasingly regarded as novel therapeutic targets. In this review, we discuss DNA alterations in CRFs and how these influence tumor chromatin structure and function, which in turn leads to tumorigenesis. We also discuss the clinical implications and review concepts of targeted treatments for these mutations. Continued research on CRF mutations will be critical for our future understanding of cancer biology and the development and implementation of novel cancer therapies. Cancer Res; 77(2); 227-33. ©2017 AACR.

Concomitant differentiation of a population of mouse embryonic stem cells into neuron‐like cells and schwann cell–like cells in a slow‐flow microfluidic device

Background: To send meaningful information to the brain, an inner ear cochlear implant (CI) must become closely coupled to as large and healthy a population of remaining spiral ganglion neurons (SGN) as possible. Inner ear gangliogenesis depends on macrophage migration inhibitory factor (MIF), a directionally attractant neurotrophic cytokine made by both Schwann and supporting cells (Bank et al., 2012). MIF‐induced mouse embryonic stem cell (mESC)‐derived “neurons” could potentially substitute for lost or damaged SGN. mESC‐derived “Schwann cells” produce MIF, as do all Schwann cells (Huang et al., a; Roth et al., 2007; Roth et al., 2008) and could attract SGN to a “cell‐coated” implant. Results: Neuron‐ and Schwann cell–like cells were produced from a common population of mESCs in an ultra‐slow‐flow microfluidic device. As the populations interacted, “neurons” grew over the “Schwann cell” lawn, and early events in myelination were documented. Blocking MIF on the Schwann cell side greatly reduced directional neurite outgrowth. MIF‐expressing “Schwann cells” were used to coat a CI: Mouse SGN and MIF‐induced “neurons” grew directionally to the CI and to a wild‐type but not MIF‐knockout organ of Corti explant. Conclusions: Two novel stem cell–based approaches for treating the problem of sensorineural hearing loss are described. Developmental Dynamics 246:7–27, 2017.

IDH1R132H acts as a tumor suppressor in glioma via epigenetic upregulation of the DNA damage response

One sentence summary Mutant IDH1 acts as a tumor suppressor when co-expressed together with TP53 and ATRX inactivating mutations in glioma, inducing genomic stability, DNA repair and resistance to genotoxic therapies. Abstract Glioma patients whose tumors carry a mutation in the Isocitrate Dehydrogenase 1 (IDH1R132H) gene are younger at the time of diagnosis and survive longer. The molecular glioma subtype which we modelled, harbors IDH1R132H, tumor protein 53 (TP53) and alpha-thalassemia/mental retardation syndrome X-linked (ATRX) loss. The impact of IDH1R132H on genomic stability, DNA damage response (DDR) and DNA repair in this molecular glioma subtype is unknown. We discovered that IDH1R132H expression in the genetic context of ATRX and TP53 inactivation: (i) increases median survival (MS), (ii) enhances DDR activity via epigenetic upregulation of Ataxia-telangiectasia mutated (ATM) signaling, and (iii) elicits tumor radioresistance. Pharmacological inhibition of ATM or checkpoint kinase 1 and 2 (CHK1/2), two essential kinases in the DDR pathways, restored tumors’ radiosensitivity. Translation of these findings for mlDH1 glioma patients could significantly improve the therapeutic efficacy of radiotherapy, and thus have a major impact on patient survival.

Native Chromatin Immunoprecipitation Using Murine Brain Tumor Neurospheres

Epigenetic modifications may be involved in the development and progression of glioma. Changes in methylation and acetylation of promoters and regulatory regions of oncogenes and tumor suppressors can lead to changes in gene expression and play an important role in the pathogenesis of brain tumors. Native chromatin immunoprecipitation (ChIP) is a popular technique that allows the detection of modifications or other proteins tightly bound to DNA. In contrast to cross-linked ChIP, in native ChIP, cells are not treated with formaldehyde to covalently link protein to DNA. This is advantageous because sometimes crosslinking may fix proteins that only transiently interact with DNA and do not have functional significance in gene regulation. In addition, antibodies are generally raised against unfixed peptides. Therefore, antibody specificity is increased in native ChIP. However, it is important to keep in mind that native ChIP is only applicable to study histones or other proteins that bind tightly to DNA. This protocol describes the native chromatin immunoprecipitation on murine brain tumor neurospheres.

Mutant ATRX: uncovering a new therapeutic target for glioma

ABSTRACT Introduction: ATRX is a chromatin remodeling protein whose main function is the deposition of the histone variant H3.3. ATRX mutations are widely distributed in glioma, and correlate with alternative lengthening of telomeres (ALT) development, but they also affect other cellular functions related to epigenetic regulation. Areas covered: We discuss the main molecular characteristics of ATRX, from its various functions in normal development to the effects of its loss in ATRX syndrome patients and animal models. We focus on the salient consequences of ATRX mutations in cancer, from a clinical to a molecular point of view, focusing on both adult and pediatric glioma. Finally, we will discuss the therapeutic opportunities future research perspectives. Expert opinion: ATRX is a major component of various essential cellular pathways, exceeding its functions as a histone chaperone (e.g. DNA replication and repair, chromatin higher-order structure regulation, gene transcriptional regulation, etc.). However, it is unclear how the loss of these functions in ATRX-null cancer cells affects cancer development and progression. We anticipate new treatments and clinical approaches will emerge for glioma and other cancer types as mechanistic and molecular studies on ATRX are only just beginning to reveal the many critical functions of this protein in cancer.

Abstract A096: Myeloid cells are required for pancreatic carcinogenesis and PD-1/PD-L1 checkpoint activation

Myeloid cells, including both macrophages and immature myeloid cells/myeloid derived suppressor cells (MDSCs), accumulate during the progression of pancreatic cancer. The goal of this study was to determine the effect of myeloid cell depletion on the onset and progression of pancreatic cancer, and to understand the relationship between myeloid cells and T cell-mediated immunity within the pancreatic cancer microenvironment. Primary mouse pancreatic cancer cells were transplanted into CD11b-DTR mice. Alternatively, the iKras* mouse model of pancreatic cancer was crossed into CD11b-DTR mice. CD11b+ cells were depleted by Diphtheria Toxin treatment during tumor initiation or in established tumors. Depletion of myeloid cells prevented KrasG12D driven pancreatic cancer initiation. Myeloid cells are required for sustained MAPK signaling in pancreatic epithelial cells during the onset of carcinogenesis, notwithstanding the expression of oncogenic Kras. In pre-established tumors, myeloid cell depletion arrested tumor growth and in some cases, induced tumor regressions that were dependent on CD8+ T cells. We found that myeloid cells inhibited CD8+ T cell anti-tumor activity by inducing the expression of Programmed cell death-ligand 1 (PD-L1) in tumor cells in an EGFR/MAPK dependent manner. Treatment with MEK inhibitors lowers the intratumoral expression of PD-L1 and renders the tumor susceptible to PD-1 blockade. Our results show that myeloid cells support immune evasion in pancreatic cancer through EGFR/MAPK dependent regulation of PD-L1 expression on tumor cells. Derailing this cross-talk between myeloid cells and tumor cells is sufficient to restore anti-tumor immunity mediated by CD8+ T cells, a finding with implications for the design of immune therapies for pancreatic cancer. Note: This abstract was not presented at the conference. Citation Format: Yaqing Zhang, Ashley Velez-Delgado, Esha Mathew, Dongjun Li, Flor M. Mendez, Kevin Flannagan, Andrew D. Rhim, Diane M. Simeone, Gregory L. Beatty, Marina Pasca di Magliano. Myeloid cells are required for pancreatic carcinogenesis and PD-1/PD-L1 checkpoint activation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A096.

Abstract 3009: ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM

Pediatric Glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most children with this diagnosis will not survive longer than two years. ATRX is a histone chaperone protein that is mutated primarily in pediatric patients with GBM and younger adults with secondary GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced in neonatal mice by injecting plasmids encoding SB transposase/ luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human glioblastoma. The loss of ATRX was specifically localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p = 0.012). ATRX-deficient tumors displayed evidence of telomeric lengthening using telomeric FISH assay for alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to develop microsatellite instability (p = 0.014), a hallmark of impaired DNA-damage repair. Analysis of three human GBM sequencing datasets confirmed increased number of somatic nucleotide mutations in ATRX-deficient tumors. Treatment of primary cell cultures generated from mouse GBMs showed that ATRX-deficient tumor cells are significantly more sensitive to certain DNA damaging agents, with greater evidence of double-stranded DNA breakage, by gH2A.X. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation showed reduced tumor growth by luminescence, with some long-term survivors. In summary, this mouse model prospectively validates ATRX as a tumor suppressor in human GBM for the first time in an animal model. In addition, loss of ATRX leads to increased genetic instability and response to DNA-damaging therapy. Based on these results, we have generated the hypothesis that ATRX loss leads to a genetically unstable tumor; which is more aggressive when untreated, but more responsive to DNA-damaging therapy, ultimately resulting in equivalent or improved overall survival. Supported by St. Baldrick9s Fellowship and Alex9s Lemonade Stand /Northwest Mutual Young Investigator Award to CK and NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Daniel Thomas, Felipe J. Nunez, Marta Dzaman, Johnny Krasinkiewicz, Rosie Lemons, Neha Kamran, Flor Mendez, Soyeon Roh, David Ferguson, Pedro R. Lowenstein, Maria G. Castro. ATRX validated as tumor suppressor in a novel mouse model of pediatric and young adult GBM. [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 3009. doi:10.1158/1538-7445.AM2015-3009

Abstract IA24: Oncogenic Kras and the pancreatic cancer microenvironment

Oncogenic mutations in the KRAS gene are almost invariably associated to pancreatic cancer in humans. Genetic engineered mice that express oncogenic Kras in the pancreas develop pancreatic cancer in a step-wise manner that resembles the progression of the human disease. Invasive cancer is preceded by Pancreatic Intraepithelial Neoplasia (PanIN) formation. In humans and in mice, pancreatic cancer and PanINs are associated to extensive accumulation of fibro-inflammatory stroma. The interactions between epithelial cells and individual components of the stroma, as well as the interaction among components of the stroma are not fully understood. We have recently identified mesenchymal stromal cells (MSCs) as one of the components of the PanIN stroma. MSCs are also present, albeit at lower frequency, in the normal pancreas. Interestingly, PanIN-derived MSCs have increased tumor-promoting ability compared to normal-pancreas-derived MSCs. MSCs have been shown to regulate the recruitment of macrophages in other tumor types. Accordingly, we find that MSCs promote macrophage recruitment in vivo. Moreover, both normal-pancreas derived MSCs and PanIN-derived MSCs promote differentiation of macrophages from bone marrow progenitors in vitro. PanIN-derived MSCs have the unique ability to promote polarization to M2- macrophages. Finally, depletion of macrophages blocks the tumor-promoting effect of MSCs in co-transplantation experiments with pancreatic cancer cells. These preliminary data our data, and previous studies in the literature, provided the rationale for an investigation of the role of monocytes-macrophages in the pancreatic cancer microenvironment. Therefore, we crossed the iKras* mouse model of pancreatic cancer with CD11b-DTR mice that allow depletion of all CD11b+ lineages (monocytes and macrophages) upon administration of Diphtheria Toxin (DT). The iKras* mouse allows inducible and reversible expression of oncogenic Kras. Expression of oncogenic Kras and induction of acute pancreatitis leads to PanIN formation and accumulation of a stroma rich in macrophages. The polarization status of these macrophages is largely M2. Inactivation of oncogenic Kras reverses the accumulation of macrophages, indicating that macrophage infiltration requires signals derived from oncogenic Kras-expressing epithelial cells (directly, or mediated by other cell types within the stroma such as MSCs or fibroblasts). Depletion of macrophages prevents PanIN formation; depletion of macrophages after PanINs have formed leads to their regression. Moreover, macrophage depletion prevents tumor establishment and causes arrest or tumor growth or regression in pre-established transplanted tumors. Thus macrophages emerge as key cell components within the pancreatic cancer stroma. We are currently investigating the mechanism underlying the requirement for macrophages during pancreatic carcinogenesis. Citation Format: Esha Mathew, Yaqing Zhang, Flor Mendez, Fil Bednar, Marina Pasca di Magliano. Oncogenic Kras and the pancreatic cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA24.